Focused libraries of genetic packages

ABSTRACT

Focused libraries of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of antibody peptides, polypeptides or proteins and collectively display, display and express, or comprise at least a portion of the focused diversity of the family. The libraries have length and sequence diversities that mimic that found in native human antibodies.

This application claims the benefit under 35 USC §120 of U.S. provisional application 60/256,380, filed Dec. 18, 2001. The provisional application and the Tables attached to it are specifically incorporated by reference herein.

The present invention relates to focused libraries of genetic packages that each display, display and express, or comprise a member of a diverse family of peptides, polypeptides or proteins and collectively display, display and express, or comprise at least a portion of the focused diversity of the family. The focused diversity of the libraries of this invention comprises both sequence diversity and length diversity. In a preferred embodiment, the focused diversity of the libraries of this invention is biased toward the natural diversity of the selected family. In a more preferred embodiment, the libraries are biased toward the natural diversity of human antibodies and are characterized by variegation in their heavy chain and light chain complementarity determining regions (“CDRs”).

The present invention further relates to vectors and genetic packages (e.g., cells, spores or viruses) for displaying, or displaying and expressing a focused diverse family of peptides, polypeptides or proteins. In a preferred embodiment the genetic packages are filamentous phage or phagemids or yeast. Again, the focused diversity of the family comprises diversity in sequence and diversity in length.

The present invention further relates to methods of screening the focused libraries of the invention and to the peptides, polypeptides and proteins identified by such screening.

BACKGROUND OF THE INVENTION

It is now common practice in the art to prepare libraries of genetic packages that individually display, display and express, or comprise a member of a diverse family of peptides, polypeptides or proteins and collectively display, display and express, or comprise at least a portion of the amino acid diversity of the family. In many common libraries, the peptides, polypeptides or proteins are related to antibodies (e.g., single chain Fv (scFv), Fv, Fab, whole antibodies or minibodies (i.e., dimers that consist of V_(H) linked to V_(L))). Often, they comprise one or more of the CDRs and framework regions of the heavy and light chains of human antibodies.

Peptide, polypeptide or protein libraries have been produced in several ways in the prior art. See e.g., Knappik et al., J. Mol. Biol., 296, pp. 57-86 (2000), which is incorporated herein by references. One method is to capture the diversity of native donors, either naive or immunized. Another way is to generate libraries having synthetic diversity. A third method is a combination of the first two. Typically, the diversity produced by these methods is limited to sequence diversity, i.e., each member of the library differs from the other members of the family by having different amino acids or variegation at a given position in the peptide, polypeptide or protein chain. Naturally diverse peptides, polypeptides or proteins, however, are not limited to diversity only in their amino acid sequences. For example, human antibodies are not limited to sequence diversity in their amino acids, they are also diverse in the lengths of their amino acid chains.

For antibodies, diversity in length occurs, for example, during variable region rearrangements. See e.g., Corbett et al., J. Mol. Biol., 270, pp. 587-97 (1997). The joining of V genes to J genes, for example, results in the inclusion of a recognizable D segment in CDR3 in about half of the heavy chain antibody sequences, thus creating regions encoding varying lengths of amino acids. The following also may occur during joining of antibody gene segments: (i) the end of the V gene may have zero to several bases deleted or changed; (ii) the end of the D segment may have zero to many bases removed or changed; (iii) a number of random bases may be inserted between V and D or between D and J; and (iv) the 5′ end of J may be edited to remove or to change several bases. These rearrangements result in antibodies that are diverse both in amino acid sequence and in length.

Libraries that contain only amino acid sequence diversity are, thus, disadvantaged in that they do not reflect the natural diversity of the peptide, polypeptide or protein that the library is intended to mimic. Further, diversity in length may be important to the ultimate functioning of the protein, peptide or polypeptide. For example, with regard to a library comprising antibody regions, many of the peptides, polypeptides, proteins displayed, displayed and expressed, or comprised by the genetic packages of the library may not fold properly or their binding to an antigen may be disadvantaged, if diversity both in sequence and length are not represented in the library.

An additional disadvantage of prior art libraries of genetic packages that display, display and express, or comprise peptides, polypeptides and proteins is that they are not focused on those members that are based on natural occurring diversity and thus on members that are most likely to be functional. Rather, the prior art libraries, typically, attempt to include as much diversity or variegation at every amino acid residue as possible. This makes library construction time-consuming and less efficient than possible. The large number of members that are produced by trying to capture complete diversity also makes screening more cumbersome than it needs to be. This is particularly true given that many members of the library will not be functional.

SUMMARY OF THE INVENTION

One objective of this invention is focused libraries of vectors or genetic packages that encode members of a diverse family of peptides, polypeptides or proteins wherein the libraries encode populations that are diverse in both length and sequence. The diverse length comprising components that contain motifs that are likely to fold and function in the context of the parental peptide, polypeptide or protein.

Another object of this invention is focused libraries of genetic packages that display, display and express, or comprise a member of a diverse family of peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the focused diversity of the family. These libraries are diverse not only in their amino acid sequences, but also in their lengths. And, their diversity is focused so as to more closely mimic or take into account the naturally-occurring diversity of the specific family that the library represents.

Another object of this invention is diverse, but focused, populations of DNA sequences encoding peptides, polypeptides or proteins suitable for display or display and expression using genetic packages (such as phage or phagemids) or other regimens that allow selection of specific binding components of a library.

A further object of this invention is focused libraries comprising the CDRs of human antibodies that are diverse in both their amino acid sequence and in their length (examples of such libraries include libraries of single chain Fv (scFv), Fv, Fab, whole antibodies or minibodies (i.e., dimers that consist of V_(H) linked to V_(L))). Such regions may be from the heavy or light chains or both and may include one or more of the CDRs of those chains. More preferably, the diversity or variegation occurs in all of the heavy chain and light chain CDRs.

It is another object of this invention to provide methods of making and screening the above libraries and the peptides, polypeptides and proteins obtained in such screening.

Among the preferred embodiments of this invention are the following:

1. A focused library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, the vectors or genetic packages being characterized by variegated DNA sequences that encode a heavy chain CDR1 selected from the group consisting of:

-   -   (1)<1>₁Y₂<1>₃M₄<1>₅, wherein <1> is an equimolar mixture of each         of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R,         S, T; V, W, and Y;     -   (2) (S/T)₁(S/G/X)₂(S/G/X)₃Y₄Y₅W₆(S/G/X)₇, wherein (S/T) is a 1:1         mixture of is and T residues, (S/G/X) is a mixture of 0.2025 S,         0.2025 G and 0.035 of each of amino acid residues A, D, E, F, H,         I, K, L, M, N, P, Q, R, T, V, W, and Y;     -   (3) V₁S₂G₃G₄S₅I₆S₇<1>₁₀<1>₉<1>₁₀Y₁₁Y₁₂W₁₃<1>₁₄, wherein <1> is         an equimolar mixture of each of amino acid residues A, D, E, F,         G, H, I, K, L, M, N, P, Q, R, S, I, V, W, and Y; and     -   (4) mixtures of vectors or genetic packages characterized by any         of the above DNA sequences, preferably in the ratio: HC CDR1s         (1):(2):(3)::0.80:0.17:0:02.

2. A focused library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody facility, the vectors or genetic packages being characterized by variegated DNA sequences that encode a heavy chain CDR2 selected from the group consisting of:

-   -   (1)<2>I<2><3>SGG<1>T<1>YADSVKG, wherein <1> is an equimolar         mixture of each of amino acid residues A, D, E, F, G, H, I, K,         L, M, N, P, Q, R, S, T, V, W, and Y; <2> is an equimolar mixture         of each of amino acid residues Y, R, W, V, G, and S; and <3> is         an equimolar mixture of each of amino acid residues P, S, and G         or an equimolar mixture of P and S;     -   (2) <1>I<4><1><1><G><5><1><1><1>YADSVKG, wherein <1> is an         equimolar mixture of each of amino acid residues A, D, E, F, G,         H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; <4> is an         equimolar mixture of residues D, I, N, S, W, Y; and <5> is an         equimolar mixture of residues S, G, D and N;     -   (3) <1>I<4><1><1>G<5><1><1>YNPSLKG, wherein <1> is an equimolar         mixture of each of amino acid residues A, D, E, F, G, H, I, K,         L, M, N, P, Q, R, S, T, V, W and Y; and <4> and <5> are as         defined above;

4)<1>I<8>S<1><1><1>GGYY<1>YAASVKG, wherein <1> is an equimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; <8> is 0.27 R and 0.027 of each of ADEFGHIKLMNPQSTVWY; and

-   -   (5) mixtures of vectors or genetic packages characterized by any         of the above DNA sequences, preferably in the ratio: HC CDR2s:         (1)/(2) (equimolar): (3):(4)::0.54:0.43:0.03.

3. A focused library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, the vectors or genetic packages being characterized by variegated DNA sequences that encode a heavy chain CDR3 selected from the group consisting of:

-   -   (1) YYCA21111YFDYWG, wherein 1 is an equimolar mixture of each         amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,         T, V; W and Y; and 2 is an equimolar mixture of K and R;     -   (2) YYCA2111111YFDYWG, wherein 1 is an equimolar mixture of each         amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,         T, V, W and Y; and 2 is an equimolar mixture of K and R;     -   (3) YYCA211111111YFDAYTG, wherein 1 is an equimolar mixture of         each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q,         R, S, T, V, W and Y; and 2 is an equimolar mixture of K and R;     -   (4) YYCAR111S2S3111YFDYWG,wherein 1 is an equimolar mixture of         each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q,         R, S, T, V, W and Y; and 2′ is an equimolar mixture of S and G;         and 3 is an equimolar mixture of Y and W;     -   (5) YYCA2111CSG11CY1YFDYWG, wherein 1 is an equimolar mixture of         each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q,         R, S, T, V, W and Y; and 2 is an equimolar mixture of K and R;     -   (6) YYCA211S1TIFG11111YFDYWG, wherein 1 is an equimolar mixture         of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P,         Q, R, S, T, V, W and Y; and 2 is an equimolar mixture of K and         R;     -   (7) YYCAR111YY2S334411.1YFDYWG, wherein 1 is an equimolar         mixture of each amino acid residues A, D, E, F, G, H, I, K, L,         M, N, P, Q, R, S, T, V, W and Y; 2 is an equimolar mixture of D         and S; and 3 is an equimolar mixture of S and G;     -   (8) YYCAR1111YC2231CY111YFDYWG, wherein 1 is an equimolar         mixture of each amino acid residues A, D, E, F, G, H, I, K, L,         M, N, P, Q, R, S, T, V, W and Y; 2 is an equimolar mixture of S         and G; and 3 is an equimolar mixture of T, D and G; and     -   (9) mixtures of vectors or genetic packages characterized by any         of the above DNA sequences, preferably the HC CDR3s (1)         through (8) are in the following proportions in the mixture:     -   (1) 0.10     -   (2) 0.14     -   (3) 0.25     -   (4) 0.13     -   (5) 0.13     -   (6) 0.11     -   (7) 0.04 and     -   (8) 0.10; and more preferably the HC CDR3s (1) through (8) are         in the following proportions in the mixture:     -   (1) 0.02     -   (2) 0.14     -   (3) 0.25     -   (4) 0.14     -   (5) 0.14     -   (6) 0.12     -   (7) 0.08 and     -   (8) 0.11.

Preferably, 1 in one or all of HC CDR3s (1) through (8) is 0.095 of each of G and Y and 0.048 of each of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, and W.

4. A focused library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, the vectors or genetic packages being characterized by variegated DNA sequences that encodes a kappa light chain CDR1 selected from the group consisting of:

-   -   (1) RASQ<1>V<2><2><3>LA     -   (2) RASQ<1>V<2><2><2><3>LA;         wherein <1> is an equimolar mixture of amino acid residues         ADEFGHIKLMNPQRSTVWY; <2> is 0.2 S and 0.044 of each of         ADEFGHIKLMNPQRTVWY; and <3> is 0.2Y and 0.044 each of         ADEFGHIKLMNPQRTVW and Y; and     -   (3) mixtures of vectors or genetic packages characterized by any         of the above DNA sequences, preferably in the ratio CDR1s         (1):(2)::0.68:0.32.

5. A focused library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, the vectors or genetic packages being characterized by variegated DNA sequences that encode a kappa light chain CDR2 having the sequence:

-   -   <1>AS<2>R<4><1>,         wherein <1> is an equimolar mixture of amino acid residues         ADEFGHIKLMNPQRSTVWY; <2> is 0.2 S and 0.044 of each of         ADEFGHIKLMNPQRTVWY; and <4> is 0.2 A and)0.044 each of         DEFGHIKLMNPQRSTVWY.

6. A focused library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, the vectors or genetic packages being characterized by variegated DNA sequences that encode a kappa light chain CDR3 selected from the groups consisting of:

-   -   (1) QQ<3><1><1><1>P<1>T,         wherein <1> is an equimolar mixture of amino acid residues         ADEFGHIKLMNPQRSTVWY; <3> is 0.2 Y and 0.044 each of         ADEFGHIKLMNPQRTVW;     -   (2) QQ33111P, wherein 1 and 3 are as defined in (1) above;     -   (3) QQ3211PP1T, wherein 1 and 3 are as defined in (1) above and         2 is 0:2 S and 0.044 each of ADEFGHIKLMNPQRTVWY; and     -   (4) mixtures of vectors or genetic packages characterized by any         of the above DNA sequences, preferably in the ratio CDR3s         (1):(2):(3)::0.65:0.1:0.25.

7. A focused library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, the vectors or genetic packages being characterized by variegated DNA sequences that encode a lambda light chain CDR1 selected from the group consisting of:

-   -   (1) TG<1>SS<2>VG<1><3><2><3>VS,         wherein <1> is 0.27 T, 0.27 G and 0.027 each of         ADEFHIKLMNPQRSVWY, <2> is 0.27 D, 0.27N and 0.027 each of         AEFGHIKLMPQRSTVWY, and <3> is 0.36 Y and 0.036 each of         ADEFGHIKLMNPQRSTVW;     -   (2) G<2><4>L<4><4><4><3><4><4>,         wherein <2> is as defined in (1) above and <4> is an equimolar         mixture of amino acid residues ADEFGHIKLMNPQRSTVWY; and     -   (3) mixtures of vectors or genetic packages characterized by any         of the above DNA sequences, preferably in the ratio CDR1s         (1):(2)::0.67:0.33.

8. A focused library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, the vectors or genetic packages being characterized by variegated DNA sequences that encode a lambda light chain CDR2 has the sequence:

-   -   <4><4><4><2>RPS,         wherein <2> is 0.27 D, 0.27 N, and 0.027 each of         AEFGHIKLMPQRSTVWY and <4> is an equimolar mixture of amino acid         residues ADEFGHIKLMNPQRSTVW.

9. A focused library of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of human antibody related peptides, polypeptides and proteins and collectively display, display and express, or comprise at least a portion of the diversity of the antibody family, the vectors or genetic packages being characterized by variegated DNA sequences that encode a lambda light chain CDR3 selected from the group consisting of:

-   -   (1)<4><5><4><2><4>S<4><4><4><4>V,         wherein <2> is 0.27 D, 0.27 N, and 0.027 each of         AEFGHIKLMPQRSTVWY; <4> is an equimolar mixture of amino acid         residues ADEFGHIKLMNPQRSTVW; and <5> is 0.36 S and 0.0355 each         of ADEFGHIKLMNPQRTVWY;     -   (2)<5>SY<1><5>S<5><1><4>V, wherein <1> is an equimolar mixture         of ADEFGHIKLMNPQRSTVWY; and <4> and <5> are as defined in (1)         above; and     -   (3) mixtures of vectors or genetic packages characterized by any         of the above DNA sequences, preferably in the ratio CDR3s

10. A focused library comprising variegated DNA sequences that encode a heavy chain CDR selected from the group consisting of:

-   -   (1) one or more of the heavy chain CDR1s of paragraph 1 above;     -   (2) one or more of the heavy chain CDR2s of paragraph 2 above;     -   (3) one or more of the heavy chain CDR3s of paragraph 3 above;         and     -   (4) mixtures of vectors or genetic packages characterized by         (1), (2) and (3).

11. The focused library comprising one or more of the variegated DNA sequences that encodes a heavy chain CDR of paragraphs 1, 2 and 3 and further comprising variegated DNA sequences that encodes a light chain CDR selected from the group consisting of

-   -   (1) one or more the kappa light chain CDR1s of paragraph 4;     -   (2) the kappa light chain CDR2 of paragraph 5;     -   (3) one or more of the kappa light chain CDR3s of paragraph 6;     -   (4) one or more of the kappa light chain CDR1s of paragraph 7;     -   (5) the lambda light chain CDR2 of paragraph 8;     -   (6) one or more of the lambda light chain CDR3s of paragraph 9;         and     -   (7) mixtures of vectors and genetic packages characterized by         one or more of (1) through (6).

12. A population of variegated DNA sequences as described in paragraphs 1-11 above.

13. A population of vectors comprising the variegated DNA sequences as described in paragraphs 1-11 above.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Antibodies (“Ab”) concentrate their diversity into those regions that are involved in determining affinity and specificity of the Ab for particular targets. These regions may be diverse in sequence or in length. Generally, they are diverse in both ways. However, within families of human antibodies the diversities, both in sequence and in length, are not truly random. Rather, some amino acid residues are preferred at certain positions of the CDRs and some CDR lengths are preferred. These preferred diversities account for the natural diversity of the antibody family.

According to this invention, and as more fully described below, libraries of vectors and genetic packages that more closely mirror the natural diversity, both in sequence and in length, of antibody families, or portions thereof are prepared and used.

Human Antibody Heavy Chain Sequence and Length Diversity

(a) Framework

The heavy chain (“HC”) Germ-Line Gene (GLG) 3-23 (also known as VP-47) accounts for about 12% of all human Abs and is preferred as the framework in the preferred embodiment of the invention. It should, however, be understood that other well-known frameworks, such as 4-34, 3-30, 3-30.3 and 4-30.1, may also be used without departing from the principles of the focused diversities of this invention.

In addition, JH4 (YFDYWGQGTLVTUSS) occurs more often than JH3 in native antibodies. Hence, it is preferred for the focused libraries of this invention. However, JH3 (AFDIWGQGTMVTVSS) could as well be used.

(b) Focused Length Diversity: CDR1, 2 and 3

(i) CDR1

For CDR1, GLGs provide CDR1s only Of the lengths 5, 6, and 7. Mutations during the maturation of the domain gene, however, can lead to CDR's having lengths as short as 2 and as long as 16. Nevertheless, length 5, predominates. Accordingly, in the preferred embodiment of this invention, the preferred HC CDR1 is 5 amino acids, with less preferred CDR1s having lengths of 7 and 14. In the most preferred libraries of this invention, all three lengths are used in proportions similar to those found in natural antibodies.

(ii) CDR2

GLGs provide CDR2s only of the lengths 15-19, but mutations during maturation may result in CDR2s of lengths from 16 to 28 amino acids. The lengths 16 and 17 predominate in mature Ab genes. Accordingly, length 17 is the preferred length for HC CDR2 of the present invention. Less preferred HC CDR2s of this invention have lengths 16 and 19. In the most preferred focused libraries of this invention, all three lengths are included in proportions similar to those found in natural antibody families.

(iii) CDR3

HC CDR3s vary in length. About half of human HCs consist of the components: V::nz::D::ny::JHn where V is a V gene, nz is a series of bases (mean 12) that are essentially random, D is a D segment, often with heavy editing at both ends, ny is a series of bases (mean 6) that are essentially random, and JH is one of the six JH segments, often with heavy editing at the 5′ end. The D segments appear to provide spacer segments that allow folding of the IgG. The greatest diversity is at the junctions of V with D and of D with JH.

In the preferred-libraries of this invention both types of HC CDR3s are used. In HC CDR3s that have no identifiable D segment, the structure is V::nz::JHn where JH is usually edited at the 5′ end. In HC CDR3s that have an identifiable D segment, the structure is V::nz::D::ny::JHn.

(c) Focused Sequence Diversity: CDR1, 2 and 3

(i) CDR1

In 5 amino acid length CDR1, examination of a 3D model of a humanized Ab showed that the side groups of residues 1, 3, and 5 were directed toward the combining pocket. Consequently, in the focused libraries of this invention, each of these positions may be selected from any of the native amino acid residues, except cysteine (“C”). Cysteine can form disulfide bonds, which are an important component of the canonical Ig fold. Having free thiol groups could, thus, interfere with proper folding of the HC and could lead to problems in production or manipulation of selected Abs. Thus, in the focused libraries of this invention cysteine is excluded from positions 1, 3 and 5 of the preferred 5 amino acid CDR1s. The other 19 natural amino acids residues may be used at positions 1, 3 and 5. Preferably, each is present in equimolar ratios in the variegated libraries of this invention.

3D modeling also suggests that the side groups of residue 2 in a 5 amino acid CDR1 are directed away from the combining pocket. Although this position shows substantial diversity, both in GLG and mature genes, in the focused libraries of this invention this residue is preferably Tyr (Y) because it occurs in 681/820 mature antibody genes. However, any of the other native amino acid residues, except Cys (C), could also be used at this position.

For position 4, there is also some diversity in GLG and mature antibody genes. However, almost all mature genes have uncharged hydrophobic amino acid residues: A, G, L, P, F, M, W, I, V, at this position. Inspection of a 3D model also shows that the side group of residue 4 is packed into the innards of the HC. Thus, in the preferred embodiment of this invention which uses framework 3-23, residue 4 is preferably Met because it is likely to fit very well into the framework of 3-23. With other frameworks, a similar fit consideration is used to assign residue 4.

Thus, the most preferred HC CDR1 of this invention consists of the amino acid sequence <1>Y<1>M<1> where <1> can be any one of amino acid residues: A, D, E, F, G, H, I, K, I, M, N, P, Q, R, S, T, V, W, Y (not C), preferably present at each position in an equimolar amount. This diversity is shown in the context of a framework 3-23:JH4 in Table 1. It has a diversity of 6859-fold.

The two less preferred HC CDR1s of this invention have length 7 and length 14. For length 7, a preferred variegation is (S/T)₁(S/G/<1>)₂(S/G/<1>)₃Y₄Y₅W₆(S/G/<1>)₇; where (S/T) indicates an equimolar mixture of Ser and Thr codons; (S/G/<1>) indicates a mixture of 0.2025 S, 0.2025 G, and 0.035 for each of A, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, Y. This design gives a predominance of Ser and Gly at positions 2, 3, and 7, as occurs in mature HC genes. For length 14, a preferred variegation is VSGGSIS<1><1><1>YYW<1>, where <1> is an equimolar mixture of the 19 native amino acid residues, except Cys (C).

The DNA that encodes these preferred HC CDR1s is preferably synthesized using trinucleotide building blocks so that each amino acid residue is present in essentially equimolar or other described amounts. The preferred codons for the <1> amino acid residues are gct, gat, gag, ttt, ggt, cat, att, aag, ctt, atg, aat, cct, cag, cgt, tct, act, gtt, tgg, and tat. Of course, other codons for the chosen amino acid residue could also be used.

The diversity oligonucleotide (ON) is preferably synthesized from BspEI to BstXI (as shown in Table 1) and can, therefore, be incorporated either by PCR synthesis using overlapping ONs or introduced by ligation of BspEI/BstXI-cut fragments. Table 2 shows the oligonucleotides that embody the specified variegations of the preferred length 5 HC CDR1s of this invention. PCR using ON-R1V1vg, ON-Ritop, and ON-R1bot gives a dsDNA product of 73 base pairs, cleavage with BspEI and BstXI trims 11 and 13 bases from the ends and provides cohesive ends that can be ligated to similarly cut vector having the 3-23 domain shown in Table 1. Replacement of ON-R1V1vg with either ONR1V2vg or ONR1V3vg (see Table 2) allows synthesis of the two alternative diversity patterns—the 7 residue length and the 14 residue length HC CDR1.

The more preferred libraries of this invention comprise the 3 preferred HC CDR1 length diversities. Most preferably, the 3 lengths should be incorporated in approximately the ratios in which they are observed in antibodies selected without reference to the length of the CDRs. For example, one sample of 1095 HC genes have the three lengths present in the ratio: L=5:L=7:L=14::820:175:23::0.80:0.17:0.02. This is the preferred ratio in accordance with this invention.

(ii) CDR2

Diversity in HC CDR2 was designed with the same considerations as for HC CDR1: GLG sequences, mature sequences and 3D structure. A preferred length for CDR2 is 17, as shown in Table 1. For this preferred 17 length CDR2, the preferred variegation in accordance with the invention is: <2>I<2><3>SGG<1>T<1>YADSVKG, where <2> indicates any amino acid residue selected from the group of Y, R, W, V, G and S (equimolar mixture), <3> is P, S and G or P and S only (equimolar mixture), and <1> is any native amino acid residue except C (equimolar mixture).

ON-R2V1vg shown in Table 3 embodies this diversity pattern. It is preferably synthesized so that fragments of dsDNA containing the BstXI and XbaI site can be generated by PCR. PCR with ON-R2V1vg, ON-R2top, and ONR2bot gives a dsDNA product of 122 base pairs. Cleavage with BstXI and XbaI removes about 10 bases from each end and produces cohesive ends that can be ligated to similarly cut vector that contains the 3-23 gene shown in Table 1.

In an alternative embodiment for a 17 length HC CDR2, the following variegation may be used: <1>I<4><1><1>G<5><1><1><1>YADSVKG, where <1> is as described above for the more preferred alternative of HC CDR2; <4> indicates an equimolar mixture of DINSWY, and <5> indicates an equimolar mixture of SGDN. This diversity pattern is embodied in ON-R2V2vg shown in Table 3. Preferably, the two embodiments are used in equimolar mixtures in the libraries of this invention.

Other preferred HC CDR2s have lengths 16 and 19. Length 16: <1>I<4><1><1>G<5<1><1>YNPSLKG; Length 19: <1>I<8>S<1><1><1>GGYY<1>YAASVKG, wherein <1> is an equimolar mixture of all native amino acid residues except C; <4> is a equimolar mixture of DINSWY; <5> is an equimolar mixture of SGDN; and <8> is 0.27 R and 0.027 of each of residues ADEFGHIKLMNPQSTVWY. Table 3 shows ON-R2V3vg which embodies a preferred CDR2 variegation of length 16 and ON-R2V4vg which embodies a preferred CDR2 variegation of length 19. To prepare these variegations ON-R2V3vg may be PCR amplified with ON-R2top and ON-R2bo3 and ON-R2V4vg may be PCR amplified with ON-R2top and ON-R2-bo4. See Table 3. In the most preferred embodiment of this invention, all three HC CDR2 lengths are used. Preferably, they are present in a ratio 17:16:19::579:464:31::0.54:0.43:0.03.

(iii) CDR3

The preferred libraries of this invention comprise several HC CDR3 components. Some of these will have only sequence diversity. Others will have sequence diversity with embedded D segments to extend the length, while also incorporating sequences known to allow Igs to fold. The HC CDR3 components of the preferred libraries of this invention and their diversities are depicted in Table 4: Components 1-8.

This set of components was chosen after studying the sequences of 1383 human HC sequences. The proposed components are meant to fulfill the following goals;

1) approximately the same distribution of lengths as seen in native Ab genes;

2) high level of sequence diversity at places having high diversity in native Ab genes; and

3) incorporation of constant sequences often seen in native Ab genes.

Component 1 represents all the genes having lengths 0 to 8 (counting from the YYCAR motif at the end of FR3 to the WG dipeptide motif near the start of the J region, i.e., FR4). Component 2 corresponds the all the genes having lengths 9 or 10. Component 3 corresponds to the genes having lengths 11 or 12 plus half the genes having length 13. Component 4 corresponds to those having length 14 plus half those having length 13. Component 5 corresponds to the genes having length 15 and half of those having length 16. Component-6 corresponds to genes of length 17 plus half of those with length 16. Component 7 corresponds to those with length 18. Component 8 corresponds to those having length 19 and greater. See Table 4.

For each HC CDR3 residue having the diversity <1>, equimolar ratios are preferably not used. Rather, the following ratios are used 0.095 [G and Y] and 0.048 [A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, and W]. Thus, there is a double dose of G and Y with the other residues being in equimolar ratios. For the other diversities, e.g., KR or SG, the residues are present in equimolar mixtures.

In the preferred libraries of this invention the eight components are present in the following fractions: 1 (0.10), 2 (0.14), 3 (0.25), 4 (0.13), 5 (0.13), 6 (0.11), 7 (0.04) and 8 (0.10). See Table 4.

In the more preferred embodiment of this invention, the amounts of the eight components is adjusted because the first component is not complex enough to justify including it as 10% of the library. For example, if the final library were to have 1×10⁹ members, then 1×10⁸ sequences would come from component 1, but it has only 2.6×10⁵ CDR3 sequences so that each one would occur in ˜385 CDR1/2 contexts. Therefore, the more preferred amounts of the eight components are 1(0.02), 2(0.14), 3(0.25), 4(0.14), 5(0.14), 6(0.12), 7(0.08), 8(0.11). In accordance with the more preferred embodiment component 1 occurs in ˜77 CDR1/2 contexts and the other, longer CDR3s occur more often.

Table 5 shows vgDNA that embodies each of the eight HC CDR3 components shown in Table 4. In Table 5, the oligonucleotides (ON) Ctop25, CtprmA, CBprmB, and CBot25 allow PCR amplification of each of the variegated ONs (vgDNA): C1t08, C2t10, C3t12, C4t14, C5t15, C6t17, C7t18, and C8t19. After amplification, the dsDNA can be cleaved with AflII and BstEII (or KpnI) and ligated to similarly cleaved vector that contains the remainder of the 3-23 domain. Preferably, this vector already contains diversity in one, or both, of CDR1 and CDR2 as disclosed herein. Most preferably, it contains-diversity in both the CDR1 and CDR2 regions. It is, of course, to be understood that the various diversities can be incorporated into the vector in any order.

Preferably, the recipient vector originally contains a stuffer in place of CDR1, CDR2 and CDR3 so that there will be no parental sequence that would then occur in the resulting library. Table 6 shows a version of the V3-23 gene segment with each CDR replaced by a short segment that contains both stop codons and restriction sites that will allow specific cleavage of any vector that does not have the stuffer removed. The stuffer can either be short and contain a restriction enzyme site that will not occur in the finished library, allowing removal of vectors that are not cleaved by both AfIII and BstEII (or KpnI) and religated. Alternatively, the stuffer could be 200-400 bases long so that uncleaved or once cleaved vector can be readily separated from doubly cleaved vector.

Human Antibody Light Chain: Sequence and Length Diversity

(i) Kappa Chain

(a) Framework

In the preferred embodiment of this invention, the kappa light chain is built in an A27 framework with a JK1 region. These are the most common V and J regions in the native genes. Other frameworks, such as O12, L2, and A11, and other J regions, such as JK4, however, may be used without departing from the scope of this invention.

(b) CDR1

In native human kappa chains, CDR1s with lengths of 11, 12, 13, 16, and 17 were observed with length 11 being predominant and length 12 being well represented. Thus, in the preferred embodiments of this invention LC CDR1s of length 11 and 12 are used in an and mixture similar to that observed in native antibodies), length 11 being most preferred. Length 11 has the following sequence: RASQ<1>V<2><2><3>LA and Length 12 has the following sequence: RASQ<1>V<25<2><2><3>LA, wherein <1> is an equimolar mixture of all of the native amino acid residues, except C, <2> is 0.2 S and 0.044 of each of ADEFGHIKLMNPQRTVWY, and <3> is 0.2 Y and 0.044 each of A, D, E, F, G, H, I, K, L, M, N, Q, R, T, V, W and Y. In the most preferred embodiment of this invention, both CDR1 lengths are used. Preferably, they are present in a ratio of 11:12::154:73:0.68:0.32.

(c) CDB2

In native kappa, CDR2 exhibits only length 7. This length is used in the preferred embodiments of this invention. It has the sequence <1>AS<2R<4><1>, wherein <1> is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY; <2> is 0.2 S and 0.004 of each of ADEFGHIKLMNPQRTVWY; and <4> is 0.2 A and 0.044 of each of DEFGHIKLMNPQRSTUWY.

(d) CDR3

In native kappa, CDR3 exhibits lengths of 1, 4, 6, 7, 8, 9, 10, 11, 12, 13, and 19. While any of these lengths and mixtures of them can be employed in this invention, we prefer lengths 8, 9 and 10, length 9 being more preferred. For the preferred Length 9, the sequence is QQ<3><1><1><1>P<1>T, wherein <1> is an equimolar mixture of amino acid residues ADEFGHIKIMNPQRSTVWY and <3> is 0.2Y and 0.044 each of ADEFGHIKLWQRSVW. Length 8 is preferably QQ33111P and Length 10 is preferably QQ3211PP1T, wherein 1 and 3 are as defined for Length 9 and 2 is S (0.2) and 0.044 each of ADEFGHIKLMNPQRTVWY. A mixture of all 3 lengths being most preferred (ratios as in native antibodies), i.e., 8:9:10::28:166:63::0.1:0.65:0.25.

Table 7 shows a kappa chain gene of this invention, including a PlacZ promoter, a ribosome-binding site, and signal sequence (M13 III signal). The DNA sequence encodes the GLG amino acid sequence, but does not comprise the GLG DNA sequence. Restriction sites are designed to fall within each framework region so that diversity can be cloned into the CDRs. XmaI and EspI are in FR1, SexAI is in FR2, RsrII is in FR3, and KpnI (or Acc65I) are in FR4. Additional sites are provided in the constant kappa chain to facilitate construction of the gene.

Table 7 also shows a suitable scheme of variegation for kappa. In CDR1, the most preferred length 11 is depicted. However, most preferably both lengths 11 and 12 are used. Length 12 in CDR1 can be construed by introducing codon 51 as <2> (i.e. a Ser-biased mixture). CDR2 of kappa is always 7 codons. Table 7 shows a preferred variegation scheme for CDR2. Table 7 shows a variegation scheme for the most preferred CDR3 (length 9). Similar variegations can be used for CDRs of length 8 and 10. In the preferred embodiment of this invention, those three lengths (8, 9 and 10) are included in the libraries of this invention in the native ratios, as described above.

Table 9 shows series of diversity oligonucleotides and primers that may be used to construct the kappa chain diversities depicted in Table 7.

(ii) Lambda Chain

(a) Framework

The lambda chain is preferably built in a 2a2 framework with an L2J region. These are the most common V and J regions in the native genes. Other frameworks, such as 3l, 4b, 1a and 6a, and other J regions, such as L1J, L3J and L7J, however, may be used without departing from the scope of this invention.

(b) CDR1

In native human lambda chains, CDR1s with length 14 predominate, lengths 11, 12 and 13 also occur. While any of these can be used in this invention, lengths 11 and 14 are preferred. For length 11 the sequence is: TG<2><4>L<4><4><4><3><4><4> and for Length 14 the sequence is: TG<1>SS<2>VG<1><3><2><3>VS, wherein <1> is 0.27 T, 0.27 G and 0.027 each of ADEFHIKLMNPQRSVWY; <2> is 0.27 D, 0.27 N and 0.027 each of AEFGHIKLMPQRSTVWY; <3> is 0.36 Y and 0.0355 each of ADEFGHIKLMNPQRSTVW; and <4> is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY. Most preferably, mixtures (similar to those occurring in native antibodies) preferably, the ratio is 11:14::23:46::0.33:0.67 of the three lengths are used.

(c) CDR2

In native human lambda chains, CDR2s with length 7 are by far the most common. This length is preferred in this invention. The sequence of this Length 7 CDR2 is <4><4><4><2>RPS, wherein <2> is 0.27 D, 0.27 N, and 0.027 each of AEFGHIKLMPQRSTVWY and <4> is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVW.

(d) CDR3

In native human lambda chains, CDR3s of length 10 and 11 predominate, while length 9 is also common. Any of these three lengths can be used in the invention. Length 11 is preferred and mixtures of 10 and 11 more preferred. The sequence of Length 11 is <4><5><4><2><4>S<4><4><4><4>V, where <2> and <4> are as defined for the lambda CDR1 and <5> is 0.36 S and 0.0355 each of ADFFGHIKLMNPQRTVWY. The sequence of Length 10 is <5>SY<1><5>S<5><1><4>V, wherein <1> is an equimolar mixture of ADEFGHIKLMNPQRSTVWY; and <4> and <5> are as defined for Length 11. The preferred mixtures of this invention comprise an equimolar mixture of Length 10 and Length 11. Table 8 shows a preferred focused lambda light chain-diversity in accordance with this invention.

Table 9 shows a series of diversity oligonucleotides and primers that may be used to construct the lambda chain diversities depicted in Table 7:

Method of Construction of the Genetic Package

The diversities of heavy chain and the kappa and lambda light chains are best constructed in separate vectors. First a synthetic gene is designed to embody each of the synthetic variable domains. The light chains are bounded by restriction sites for ApaLI (positioned at the very end of the signal sequence) and AscI (positioned afer the stop codon). The heavy chain is bounded by SfiI (positioned within the PelB signal sequence) and NotI (positioned in the linker between CH1 and the anchor protein). Signal sequences other than PelB may also need, e.g., a M13 pIII signal sequence.

The initial genes are made with “stuffer” sequences in place of the desired CDRs. A “Stuffer” is a sequence that is to be cut away and replaced by diverse DNA but which does not allow expression of a functional antibody gene. For example, the stuffer may contain several stop codons and restriction sites that will not occur in the correct finished library vector. For example, in Table 10, the stuffer for CDR1 of kappa A27 contains a StuI site. The vgDNA for CDR1 is introduced as a cassette from EspI, XmaI, or AflII to either SexAI or KasI. After the ligation, the DNA is cleaved with StuI; there should be no StuI sites in the desired vectors.

The sequences of the heavy chain gene with stuffers is depicted in Table 6. The sequences of the kappa light chain gene with stuffers is depicted in Table 10. The sequence of the lambda light chain gene with stuffers is depicted in Table 11.

In another embodiment of the present intention the diversities of heavy chain and the kappa or lambda light chains are constructed in a single vector or genetic packages (e.g., for display or display and expression) having appropriate restriction sites that allow cloning of these chains. The processes to construct such vectors are well known and widely used in the art. Preferably, a heavy chain and Kappa light chain library and a heavy chain and lambda light chain library would be prepared separately. The two libraries, most preferably, will then be mixed in equimolar amounts to attain maximum diversity.

Most preferably, the display is had on the surface of a derivative of M13 phage. The most preferred vector contains all the genes of M13, an antibiotic resistance gene, and the display cassette. The preferred vector is provided with restriction sites that allow introduction and excision of members of the diverse family of genes, as cassettes. The preferred vector is stable against rearrangement under the growth conditions used to amplify phage.

In another embodiment of this invention, the diversity captured by the methods of the present invention may be displayed and/or expressed in a phagemid vector (e.g., pCES1) that displays and/or expresses the peptide, polypeptide or protein. Such vectors may also be used to store the diversity for subsequent display and/or expression using other vectors or phage.

In another embodiment of this invention, the diversity captured by the methods of the present invention may be displayed and/or expressed in a yeast vector.

TABLE 1 3-23:JH4 CDR1/2 diversity = 1.78 × 10⁸                       FR1(VP47/V3-23)---------------            20  21  22             23  24  25  26  27  28  29  30             A   M   A              E   V   Q   L   L   E   S   G ctgtctgaac  cc atg gcc            gaa|gtt|caa|ttg|tta|gag|tct|ggt| Scab......  NcoI....                        MfeI --------------FR1--------------------------------------------  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45   G   G   L   V   Q   P   G   G   S   L   R   L   S   C   A |ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gct|     Sites of variegation      <1>      <1> <1> <1>                6859-fold diversity ----FR1-------------------->|.....CDR1....................|---FR2------  46  47  48  49  50  51  52   53  54   55  56  57  58  59  60   A   S   G   F   T   F   S   -   Y   -   M   -   W   V   R |gct|tcc|gga|ttc|act|ttc|tct| - |tac| - |atg| - |tgg|gtt|cgc|       BspEI                     BsiWI                       BstXI.                        Sites of variegation-><2>     <2> <3> -------FR2-------------------------------->|...CDR2.........  61  62  63  64  65  66  67  68  69  70  71  72  73  74  75   Q   A   P   G   K   G   L   E   W   V  S   -   I   -   - |caa|gct|cct|ggt|aaa|ggt|ttg|gag|tgg|gtt|tct| - |atc| - | - | ...BstXI               <1>     <1>  25992-fold diversity in CDR2 .....CDR2............................................|---FR3---   76  77  78  79  80  81  82  83  84  85  86  87  88  89  90    S   G   G   -   T   -   Y   A   D   S   V   K   G   R   F |tct|ggt|ggc| - |act| - |tat|gct|gac|tcc|gtt|aaa|ggt|cgc|ttc| --------FR3--------------------------------------------------   91  92  93  94  95  96  97  98  99 100 101 102 103 104 105   T   I   S   R   D   N   S   K   N   T   L   Y   L   Q   M |act|atc|tct|aga|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|          XbaI ---FR3----------------------------------------------------->|  106 107 108 109 110 111 112 113 114 115 116 117 118 119 120   N   S   L   R   A   E   D   T   A   V   Y   Y   C   A   K |aac|agc|tta|agg|gct|gag|gac|acc|gct|gtc|tac|tac|tgc|gcc|aaa|         AflII .......CDR3.................| Replaced by the various components!  121 122 123 124 125 126 127   D   Y   E   G   T   G   Y |gac|tat|gaa|ggt|act|ggt|tat| |----- FR4 ---(JH4)-----------------------------------------   Y   F   D   Y   W   G   Q   G   T   L   V   T   V   S   S |tat|ttc|gat|tat|tgg|ggt|caa|ggt|acc|ctg|gtc|acc|gtc|tct|agt|...                              KpnI       BstEII <1> = Codons for ADEFGHIKLMNPQRSTVWY (equimolar mixture) <2> = Codons for YRWVGS (equimolar mixture) <3> = Codons for PS or PS and G (equimolar mixture)

TABLE 2 Oligonucleotides used to variegate CDR1 of human HC CDR1 - 5 residues (ON-R1V1vg): 5′-ct|tcc|gga|ttc|act|ttc|tct|<1>|tac|<1>|atg|<1>|tgg|gtt|cgc|caa| gct|cct|gg-3′ <1> = Codons of ADEFGHIKLMNPQRSTVWY 1:1 (ON-R1top): 5′-cctactgtct|tcc|gga|ttc|act|ttc|tct-3′ (ON-R1bot) [RC]: 5′-tgg|gtt|cgc|caa|gct|cct|ggttgctcactc-3′ CDR1 - 7 residues (ON-R1V2vg): 5′-ct|tcc|gga|ttc|act|ttc|tct|<6>|<7>|<7>|tac|tac|tgg|<7>|tgg| gtt|cgc|caa|gct|cct|gg-3′ <6> = Codons for ST, 1:1 <7> = 0.2025(Codons for SG) + 0.035(Codons for ADEFHIKLMNPQRTVWY) CDR1 - 14 residues (ON-R1V3vg): 5′-ct|tcc|gga|ttc|act|ttc|tct|atc|agc|ggt|ggt|tct|atc|tcc|<1>|<1>|<1>|- tac|tac|tgg|<1>|tgg|gtt|cgc|caa|gct|cct|gg-3′ <1> = Codons for ADEFGHIKLMNPQRSTVWY 1:1

TABLE 3 Oligonucleotides used to variegate CDR2 of human HC CDR2 - 17 residues (ON-R2V1vg): 5′-ggt|ttg|gag|tgg|gtt|tct|<2>|atc|<2>|<3>|tct|ggt|ggc|<1>|act|<1>|tat|gct|- gac|tcc|gtt|aaa|gg-3′ (ON-R2top): 5′-ct|tgg|gtt|cgc|caa|gct|cct|ggt|aaa|ggt|ttg|gag|tgg|gtt|tct-3′ (ON-R2bot) [RC]: 5′-tat|gct|gac|tcc|gtt|aaa|ggt|cgc|ttc|act|atc|tct|aga|ttcctgtcac-3′ <1> = Codons for A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y (equimolar mixture) <2> = Codons for Y, R, W, V, G and S (equimolar mixture) <3> = Codons for P and S (equimolar mixture) or P, S and G (equimolar mixture) (ON-R2V2vg): 5′-ggt|ttg|gag|tgg|gtt|tct|<1>|atc|<4>|<1>|<1>|ggt|<5>|<1>|<1>|<1>|tat|gct|- gac|tcc|gtt|aaa|gg-3′ <4> = Codons for DINSWY (equimolar mixture) <5> = Codons for SGDN, (equimolar mixture) CDR2 - 16 residues (ON-R2V3vg): 5′-ggt|ttg|gag|tgg|gtt|tct|<1>|atc|<4>|<1>|<1>|ggt| <5>|<1>|<1>|tat|aac|cct|tcc|ctt|aag|gg-3′ (ON-R2bo3) [RC]: 5′-tat|aac|cct|tcc|ctt|aag|ggt|cgc|ttc|act|atc|tct|aga|ttcctgtcac-3′ CDR2 - 19 residues (ON-R2V4vg): 5′-ggt|ttg|gag|tgg|gtt|tct|<1>|atc|<8>|agt|<1>|<1>| <1>|ggt|ggt|act|act|<1>|tat|gcc|gct|tcc|gtt|aag|gg-3′ (ON-R2bo4) [RC]: 5′-tat|gcc|gct|tcc|gtt|aag|ggt|cgc|ttc|act|atc|tct|aga|ttcctgtcac-3′ <1>, <2>, <3>, <4> and <5> are as defined above <8> is 0.27 R and 0.027 each of ADEFGHIKLMNPQSTVWY

TABLE 4 Preferred Components of HC CDR3 Preferred Fraction Adjusted Component Length Complexity of Library Fraction 1 YYCA21111YFDYWG. 8 2.6 × 10⁵ .10 .02 (1 = any amino acid residue, except C; 2 = K and R) 2 YYCA2111111YFDYWG. 10 9.4 × 10⁷ .14 .14 (1 = any amino acid residue, except C; 2 = K and R) 3 YYCA211111111YFDYTG. 12 3.4 × 10¹⁰ .25 .25 (1 = any amino acid residue, except C; 2 = K and R) 4 YYCAR111S2S3111YFDYWG. 14 1.9 × 10⁸ .13 .14 (1 = any amino acid residue, except C; 2 = S and G 3 = Y and W) 5 YYCA2111CSG11CY1YFDYWG. 15 9.4 × 10⁷ .13 .14 (1 = any amino acid residue, except C; 2 = K and R) 6 YYCA211S1TIFG11111YFDYWG. 17 1.7 × 10¹⁰ .11 .12 (1 = any amino acid residue, except C; 2 = K and R) 7 YYCAR111YY2S33YY111YFDYWG. 18 3.8 × 10⁸ .04 .08 (1 = any amino acid residue, except C; 2 = D or G; 3 = S and G) 8 YYCAR1111YC2231CY111YFDYWG. 19 2.0 × 10¹¹ .10 .11 (1 = any amino acid residue, except C; 2 = S and G; 3 = T, D and G)

TABLE 5 Oligonucleotides used to variegate the eight components of HC CDR3 (Ctop25): 5′-gctctggtcaac|tta|agg|gct|gag|g-3′ (CtprmA): 5′-gctctggtcaac|tta|agg|gct|gag|gac|acc|gct|gtc|tac|tac|tgc|gcc-3′               AflII... (CBprmB) [RC]: 5′-|tac|ttc|gat|tac|tgg|ggc|caa|ggt|acc|ctg|gtc|acc|tcgctccacc-3′                                           BstEII... (CBot25) [RC]: 5′-|ggt|acc|ctg|gtc|acc|tcgctccacc-3′ The 20 bases at 3′ end of CtprmA are identical to the most 5′ 20 bases of each of the vgDNA molecules. Ctop25 is identical to the most 5′ 25 bases of CtprmA. The 23 most 3′ bases of CBprmB are the reverse complement of the most 3′ 23 bases of each of the vgDNA molecules. CBot25 is identical to the 25 bases at the 5′ end of CBprmB. Component 1 (C1t08): 5′-cc|gct|gtc|tac|tac|tgc|gcc|<2>|<1>|<1>|<1>|<1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3′ <1> = 0.095 Y + 0.095 G + 0.048 each of the residues ADEFHIKLMNPQRSTVW, no C; <2> = K and R (equimolar mixture) Component 2 (C2t10): 5′-cc|gct|gtc|tac|tac|tgc|gcc|<2>|<1>|<1>|<1>|<1>|<1>|<1>|tac|ttc|gat|tac|tgg| ggc|caa|gg-3′ <1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C; <2> = K and R (equimolar mixture) Component 3 (C3t12): 5′-cc|gct|gtc|tac|tac|tgc|gcc|<2>|<1>|<1>|<1>|<1>|<1>|<1>|<1>|<1>|tac|ttc|gat|tac|- tgg|ggc|caa|gg-3′ <1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C; <2> = K and R (equimolar mixture) Component 4 (C4t140): 5′-cc|gct|gtc|tac|tac|tgc|gcc|cgt|<1>|<1>|<1>|tct|<2>|tct|<3>|<1>|<1>|<1>|tac|ttc|gat|- tac|tgg|ggc|caa|gg-3′ <1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C; <2> = S and G (equimolar mixture); <3> = Y and W (equimolar mixture) Component 5 (C5t15): 5′-cc|gct|gtc|tac|tac|tgc|gcc|<2>|<1>|<1>|<1>|tgc|tct|ggt|<1>|<1>|tgc|tat|<1>|tac|- ttc|gat|tac|tgg|ggc|caa|gg-3′ <1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C; <2> = K and R (equimolar mixture) Component 6 (C6t17): 5′-cc|gct|gtc|tac|tac|tgc|gcc|<2>|<1>|<1>|tct|<1>|act|atc|ttc|ggt|<1>|<1>|<1>|<1>|- <1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3′ <1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C; <2> = K and R (equimolar mixture) Component 7 (C7t18): 5′-cc|gct|gtc|tac|tac|tgc|gcc|cgt|<1>|<1>|<1>|tat|tac|<2>|tct|<3>|<3>|tac|tat|- <1>|<1>|<1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3′ <1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C; <2> = D and G (equimolar mixture); <3> = S and G (equimolar mixture) Component 8 (c8t19): 5′-cc|gct|gtc|tac|tac|tgc|gcc|cgt|<1>|<1>|<1>|<1>|tat|tgc|<2>|<2>|<3>|<1>|tgc|tat|- <1>|<1>|<1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3′ <1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C; <2> = S and G (equimolar mixture); <3> = TDG (equimolar mixture);

TABLE 6 3-23::JH4 Stuffers in place of CDRs                                   FR1(DP47/V3-23)---------------            20  21  22             23  24  25  26  27  28  29  30             A   M   A              E   V   Q   L   L   E   S   G ctgtctgaac  cc atg gcc            gaa|gtt|caa|ttg|tta|gag|tct|ggt| Scab......  NcoI....                        MfeI --------------FR1--------------------------------------------  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45   G   G   L   V   Q   P   G   G   S   L   R   L   S   C   A |ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gct| ----FR1-------------------->|...CDR1 stuffer....|---FR2------  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   A   S   G   F   T   F   S   S   Y   A   |    |   W   V   R |gct|tcc|gga|ttc|act|ttc|tct|tcg|tac|gct|tag|taa|tgg|gtt|cgc|       BspEI                     BsiWI                       BstXI. -------FR2-------------------------------->|...CDR2 stuffer. 61  62  63  64  65  66  67  68  69  70  71  72  73  74  75  Q   A   P   G   K   G   L   E   W   V   S   |   p   r   | |caa|gct|cct|ggt|aaa|ggt|ttg|gag|tgg|gtt|tct|taa|cct|agg|tag| ...BstXI                                         AvrII.. .....CDR2 stuffer....................................|---FR3---     91  92  93  94  95  96  97  98  99 100 101 102 103 104 105     T   I   S   R   D   N   S   K   N   T   L   Y   L   Q   M |act|atc|tct|aga|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|           XbaI ---FR3-----------..> CDR3 Stuffer------------->|  106 107 108 109 110   N   S   L   R   A |aac|agc|tta|agg|gct|tag taa agg cct taa         AflII                StuI... |----- FR4 ---(JH4)-----------------------------------------  Y   F   D   Y   W   G   Q   G   T   L   V   T   V   S   S |tat|ttc|gat|tat|tgg|ggt|caa|ggt|acc|ctg|gtc|acc|gtc|tct|agt|...                               KpnI       BstEII

TABLE 7 A27:JH1 Human Kappa light chain gene gaggacc attgggcccc ctccgagact ctcgagcgca Scab...... EcoO109I           XhoI..            ApaI. acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc       ..-35..          Plac                    ..-10. cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctt tggagccttt tttttggaga ttttcaac   PflMI.......       Hind III M13 III signal sequence (AA seq)--------------------------->  1   2   3   4   5   6   7   8   9  10  11  12  13  14  15  M   K   K   L   L   F   A   I   P   L   V   V   P   F   Y gtg aag aag ctc cta ttt gct atc ccg ctt gtc gtt ccg ttt tac --Signal-->FR1------------------------------------------->  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30   S   H   S   A   Q   S   V   L   T   Q   S   P   G   T   L |agc|cat|agt|gca|caa|tcc|gtc|ctt|act|caa|tct|cct|ggc|act|ctt|           ApaLI... ----- FR1 ------------------------------------->| CDR1------>  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45   S   L   S   P   G   E   R   A   T   L   S   C   R   A   S |tcg|cta|agc|ccg|ggt|gaa|cgt|gct|acc|tta|agt|tgc|cgt|gct|tcc|    EspI.....                       AflII...            XmaI.... For CDR1: <1> ADEFGHIKLMNPQRSTVWY 1:1 <2> S(0.2) ADEFGHIKLMNPQRTVWY (0.044 each) <3> Y(0.2) ADEFGHIKLMNPQRSTVW (0.044 each) (CDR1 installed as AflII-(SexAI or KasI) cassette.) For the most preferred 11 length codon 51 (XXX) is omitted; for the preferred 12 length this codon is <2> ------- CDR1 --------------------->|--- FR2 --------------->     <1>     <2> <2> xxx <3> 46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  Q   -   V   -   -   -   -  L   A   W   Y   Q   Q   K   P |cag| - |gtt| - | - | - | - |ctt|gct|tgg|tat|caa|cag|aaa|cct|                                                       SexAI... For CDR2: <1> ADEFGHIKLMNPQRSTVWY 1:1 <2> S(0.2) ADEFGHIKLMNPQRTVWY (0.044 each) <4> A(0.2) DEFGHIKLMNPQRSTVWY (0.044 each) CDR2 installed as (SexAI or KasI) to (BamHI or RsrII) cassette.) ----- FR2 ------------------------->|------- CDR2 ---------->                                      <1>         <2>     <4>   61  62  63  64  65  66  67  68  69  70  71  72  73  74  75    G   Q   A   P   R   L   L   I   Y   -   A   S   -   R   - |ggt|cag|gcg|ccg|cgt|tta|ctt|att|tat| - |gct|tct| - |cgc| - | SexAI.... KasI.... CDR2-->|--- FR3 -----------------------------------------------> <1>  76  77  78  79  80  81  82  83  84  85  86  87  88  89  90   -   G   I   P   D   R   F   S   G   S   G   S   G   T   D | - |ggg|atc|ccg|gac|cgt|ttc|tct|ggc|tct|ggt|tca|ggt|act|gac|       BamHI...              RsrII..... ------ FR3 ------------------------------------------------->  91  92  93  94  95  96  97  98  99 100 101 102 103 104 105   F   T   L   T   I   S   R   L   E   P   E   D   F   A   V |ttt|acc|ctt|act|att|tct|aga|ttg|gaa|cct|gaa|gac|ttc|gct|gtt|                      XbaI... For CDR3 (Length 9): <1> ADEFGHIKLMNPQRSTVWY 1:1 <3> Y(0.2) ADEFGHIKLMNPQRTVW (0.044 each) For CDR3 (Length 8): QQ33111P 1 and 3 as defined for Length 9 For CDR3 (Length 10): QQ3211PP1T 1 and 3 as defined for Length 9 2 S(0.2) and 0.044 each of ADEFGHIKLMNPQRTVWY CDR3 installed as XbaI to (StyI or BsiWI) cassette. ----------->|----CDR3-------------------------->|-----FR4--->                      <3> <1> <1> <1>     <1> 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120   Y   Y   C   Q   Q   -   -   -   -   P   -   T   F   G   Q |tat|tat|tgc|caa|cag| - | - | - | - |cct| - |act|ttc|ggt|caa|            BstXI........... -----FR4------------------->|  <------- Ckappa ------------ 121 122 123 124 125 126 127    128 129 130 131 132 133 134   G   T   K   V   E   I   K     R   T   V   A   A   P   S |ggt|acc|aag|gtt|gaa|atc|aag| |cgt|acg|gtt|gcc|gct|cct|agt|       StyI....                 BsiWI.. 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149   V   F   I   F   P   P   S   D   E   Q   L   K   S   G   T |gtg|ttt|atc|ttt|cct|cct|tct|gac|gaa|caa|ttg|aag|tca|ggt|act|                                      MfeI... 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164   A   S   V   V   C   L   L   N   N   F   Y   P   R   E   A |gct|tct|gtc|gta|tgt|ttg|ctc|aac|aat|ttc|tac|cct|cgt|gaa|gct|                                               BssSI...  165 166 167 168 169 170 171 172 173 174 175 176 177 178 179   K   V   Q   W   K   V   D   N   A   L   Q   S   G   N   S |aaa|gtt|cag|tgg|aaa|gtc|gat|aac|gcg|ttg|cag|tcg|ggt|aac|agt|                               MluI....  180 181 182 183 184 185 186 187 188 189 190 191 192 193 194   Q   E   S   V   T   E   Q   D   S   K   D   S   T   Y   S |caa|gaa|tcc|gtc|act|gaa|cag|gat|agt|aag|gac|tct|acc|tac|tct|  195 196 197 198 199 200 201 202 203 204 205 206 207 208 209   L   S   S   T   L   T   L   S   K   A   D   Y   E   K   H |ttg|tcc|tct|act|ctt|act|tta|tca|aag|gct|gat|tat|gag|aag|cat|  210 211 212 213 214 215 216 217 218 219 220 221 222 223 224   K   V   Y   A   C   E   V   T   H   Q   G   L   S   S   P |aag|gtc|tat|GCt|TGC|gaa|gtt|acc|cac|cag|ggt|ctg|agc|tcc|cct|                                                SacI....  225 226 227 228 229 230 231 232 233 234   V   T   K   S   F   N   R   G   E   C   .   . |gtt|acc|aaa|agt|ttc|aac|cgt|ggt|gaa|tgc|taa|tag ggcgcgcc                        DsaI....                  AscI....                                                   BssHII acgcatctctaa gcggccgc aacaggaggag              NotI....

TABLE 8 2a2:JH2 Human lambda-chain gene gaggaccatt gggcccc   ttactccgtgac Scab...... EcoO109I            ApaI..         -----------FR1-------------------------------------------->          1   2   3   4   5   6   7   8   9  10  11  12  13  14  15  S   A   Q   S   A   L   T   Q   P   A   S   V   S   G   S   P   G agt|gca|caa|tcc|gct|ctc|act|cag|cct|gct|agc|gtt|tcc|ggg|tca|cct|ggt|  ApaLI...                           NheI...          BstEII...                                                           SexAI.... For CDR1 (length 14): <1> = 0.27 T, 0.27 G, 0.027 each of ADEFHIKLMNPQRSVWY, no C <2> = 0.27 D, 0.27 N, 0.027 each of AEFGHIKLMPQRSTVWY, no C <3> = 0.36 Y, 0.0355 each of ADEFGHIKLMNPQRSTVW, no C                               T   G  <1>  S   S  <2>  V   G ------FR1------------------> |-----CDR1---------------------  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30   Q   S   I   T   I   S   C   T   G   -   S   S   -   V   G |caa|agt|atc|act|att|tct|tgt|aca|ggt| - |tct|tct| - |gtt|ggc|                          BsrGI..  <1> <3> <2> <3>  V   S = vg Scheme #1, length = 14  -----CDR1------------->|--------FR2-------------------------  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45   -   -   -   -   V   S   W   Y   Q   Q   H   P   G   K   A | - | - | - | - |gtt|tct|tgg|tat|caa|caa|cac|ccg|ggc|aag|gcg|                                           XmaI....    KasI.....                                           AvaI.... A second Vg scheme for CDR1 gives segments of length 11: T₂₂G<2><4>L<4><4><4><3><4><4> where <4> = equimolar mixture of each of ADEFGHIKLMNPQRSTVWY, no C <3> = as defined above for the alternative CDR1 For CDR2: <2> and <4> are the same variegation as for CDR1                         <4> <4> <4> <2>  R   P   S  --FR2-----------------> |------CDR2--------------->|-----FR3-  46  47  48  49  50  51  52  53  54  55  56  57  58  59  60   P   K   L   M   I   Y   -   -   -   -   R   P   S   G   V |ccg|aag|ttg|atg|atc|tac| - | - | - | - |cgt|cct|tct|ggt|gtt| KasI.... -------FR3----------------------------------------------------  61  62  63  64  65  66  67  68  69  70  71  72  73  74  75   S   N   R   F   S   G   S   K   S   G   N   T   A   S   L |agc|aat|cgt|ttc|tcc|gga|tct|aaa|tcc|ggt|aat|acc|gca|agc|tta|                  BspEI..                           HindIII.                       BsaBI........(blunt) -------FR3-------------------------------------------------->|  76  77  78  79  80  81  82  83  84  85  86  87  88  89  90   T   I   S   G   L   Q   A   E   D   E   A   D   Y   Y   C |act|atc|tct|ggt|ctg|cag|gct|gaa|gac|gag|gct|gac|tac|tat|tgt|                  PstI... CDR3 (Length 11): <2> and <4> are the same variegation as for CDR1 <5> = 0.36 S, 0.0355 each of ADEFGHIKLMNPQRTVWY no C CDR3 (Length 10): <5> SY <1> <5> S <5> <1> <4> V <1> is an equimolar mixture of ADEFGHIKLMNPQRSTVWY, no C <4> and <5> are as defined for Length 11   <4> <5> <4> <2> <4> S <4> <4> <4> <4> V -----CDR3---------------------------------->|---FR4---------  91  92  93  94  95  96  97  98  99  100 101 102 103 104 105   -   -   -   -   -   S   -   -   -   -    V   F   G   G   G | - | - | - | - | - |tct| - | - | - | - |gtc|ttc|ggc|ggt|ggt|                                                          KpnI... -------FR4-------------->  106 107 108 109 110 111 112 113 114 115 116 117 118 119 120   T   K   L   T   V   L   G   Q   P   K   A   A   P   S   V |acc|aaa|ctt|act|gtc|ctc|ggt|caa|cct|aag|gct|gct|cct|tcc|gtt| KpnI...                   HincII..                                 Bsu36I...  121 122 123 124 125 126 127 128 129 130 131 132 133 134 135   T   L   F   P   P   S   S   E   E   L   Q   A   N   K   A |act|ctc|ttc|cct|cct|agt|tct|gaa|gag|ctt|caa|gct|aac|aag|gct|                              SapI.....  136 137 138 139 140 141 142 143 144 145 146 147 148 149 150   T   L   V   C   L   I   S   D   F   Y   P   G   A   V   T |act|ctt|gtt|tgc|ttg|atc|agt|gac|ttt|tat|cct|ggt|gct|gtt|act|                   BclI.... 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165   V   A   W   K   A   D   S   S   P   V   K   A   G   V   E |gtc|gct|tgg|aaa|gcc|gat|tct|tct|cct|gtt|aaa|gct|ggt|gtt|gag|                                                          BsmBI...  166 167 168 169 170 171 172 173 174 175 176 177 178 179 180   T   T   T   P   S   K   Q   S   N   N   K   Y   A   A   S |acg|acc|act|cct|tct|aaa|caa|tct|aac|aat|aag|tac|gct|gcg|agc| BsmBI....                                              SacI....  181 182 183 184 185 186 187 188 189 190 191 192 193 194 195   S   Y   L   S   L   T   P   E   Q   W   K   S   H   K   S |tct|tat|ctt|tct|ctc|acc|cct|gaa|caa|tgg|aag|tct|cat|aaa|tcc| SacI...  196 197 198 199 200 201 202 203 204 205 206 207 208 209 210   Y   S   C   Q   V   T   H   E   G   S   T   V   E   K   T |tat|tcc|tgt|caa|gtt|act|cat|gaa|ggt|tct|acc|gtt|gaa|aag|act|                        BspHI...  211 212 213 214 215 216 217 218 219   V   A   P   T   E   C   S   .   . |gtt|gcc|cct|act|gag|tgt|tct|tag|tga|ggcgcgcc                                     AscI....                                      BssHII aacgatgttc aag gcggccgc aacaggaggag                NotI.... Scab.......

TABLE 9 Oligonucleotides For Kappa and Lambda Light Chain Variegation (Ctop25): 5′-gctctggtcaac|tta|agg|gct|gag|g-3′ (CtprmA): 5′-gctctggtcaac|tta|agg|gct|gag|gac|acc|gct|gtc|tac|tac|tgc|gcc-3′               AflII... (CBprmB) [RC]: 5′-|tac|ttc|gat|tac|ttg|ggc|caa|ggt|acc|ctg|gtc|acc|tcgctccacc-3′                                           BstEII... (CBot25) [RC]: 5′-|ggt|acc|ctg|gtc|acc|tcgctccacc-3′ Kappa chains: CDR1 (“1”), CDR2 (“2”), CDR3 (“3”) CDR1 (Ka1Top610): 5′-ggtctcagttg|cta|agc|ccg|ggt|gaa|cgt|gct|acc|tta|agt|tgc| cgt|gct|tcc|cag-3′ (Ka1STp615): 5′-ggtctcagttg|cta|agc|ccg|ggt|g-3′ (Ka1Bot620) [RC]: 5′-ctt|gct|tgg|tat|caa|cag|aaa|cct|ggt|cag|gcg|ccaagtcgtgtc-3′ (Ka1SB625) [RC]: 5′-cct|ggt|cag|gcg|ccaagtcgtgtc-3′ (Ka1vg600): 5′-gct|acc|tta|agt|tgc|cgt|gct|tcc|cag- |<1>|gtt|<2>|<2>|<3>|ctt|gct|tgg|tat|caa|cag|aaa|cc-3′ (Ka1vg600-12): 5′-gct|acc|tta|agt|tgc|cgt|gct|tcc|cag- |<1>|gtt|<2>|<2>|<2>|<3>|ctt|gct|tgg|tat|caa|cag|aaa|cc-3′ CDR2 (Ka2Tshort657): 5′-cacgagtccta|cct|ggt|cag|gc-3′ (Ka2Tlong655): 5′-cacgagtccta|cct|ggt|cag|gcg|ccg|cgt|tta|ctt|att|tat-3′ (Ka2Bshort660): [RC]: 5′-|gac|cgt|ttc|tct|ggt|tctcacc-3′ (Ka2vg650): 5′-cag|gcg|ccg|cgt|tta|ctt|att|tat|<1>|gct|tct|<2>|-       |cgc|<4>|<1>|ggg|atc|ccg|gac|cgt|ttc|tct|ggt|tctcacc-3′ CDR3 (Ka3Tlon672): 5′-gacgagtccttct|aga|ttg|gaa|cct|gaa|gac|ttc|gct|gtt|tat|tat|tgc|caa|c-3′ (Ka3BotL682) [RC]: 5′-act|ttc|ggt|caa|ggt|acc|aag|gtt|gaa|atc|aag|cgt|acg|tcacaggtgag-3′ (Ka3Bsho694) [RC]: 5′-gaa|atc|aag|cgt|acg|tcacaggtgag-3′ (Ka3vg670): 5′-gac|ttc|gct|gtt|- |tat|tat|tgc|caa|cag|<3>|<1>|<1>|<1>|cct|<1>|act|ttc|ggt|caa|- |ggt|acc|aag|gtt|g-3′ (Ka3vg670-8): 5′-gac|ttc|gct|gtt|- |tat|tat|tgc|caa|cag|<3>|<3>|<1>|<1>|<1>|cct|ttc|ggt|caa|- |ggt|acc|aag|gtt|g-3′ (Ka3vg670-10): 5′-gac|ttc|gct|gtt|tat|- |tat|tgc|caa|cag|<3>|<2>|<1>|<1>|cct|cct|<1>|act|ttc|ggt|caa|- |ggt|acc|aag|gtt|g-3′ Lambda Chains: CDR1 (“1”), CDR2 (“2”), CDR3 (“3”) CDR1 (Lm1TPri75): 5′-gacgagtcctgg|tca|cct|ggt|-3′ (Lm1tlo715): 5′-gacgagtcctgg|tca|cct|ggt|caa|agt|atc|act|att|tct|tgt|aca|ggt-3′ (Lm1blo724) [rc]: 5′-gtt|tct|tgg|tat|caa|caa|cac|ccg|ggc|aag|gcg|agatcttcacaggtgag-3′ (Lm1bsh737) [rc]: 5′-gc|aag|gcg|agatcttcacaggtgag-3′ (Lm1vg710b): 5′-gt|atc|act|att|tct|tgt|aca|ggt|<2>|<4>|ctc|<4>|<4>|<4>|- |<3>|<4>|<4>|tgg|tat|caa|caa|cac|cc-3′ (Lm1vg710): 5′-gt|atc|act|att|tct|tgt|aca|ggt|<1>|tct|tct|<2>|gtt|ggc|-        |<1>|<3>|<2>|<3>|gtt|tct|tgg|tat|caa|caa|cac|cc-3′ CDR2 (Lm2TSh757): 5′-gagcagaggac|ccg|ggc|aag|gc-3′ (Lm2TLo753): 5′-gagcagaggac|ccg|ggc|aag|gcg|ccg|aag|ttg|atg|atc|tac|-3′ (Lm2BLo762) [RC]: 5′-cgt|cct|tct|ggt|gtc|agc|aat|cgt|ttc|tcc|gga|tcacaggtgag-3′ (Lm2BSh765) [RC]: 5′-cgt|ttc|tcc|gga|tcacaggtgag-3′ (Lm2vg750): 5′-g|ccg|aag|ttg|atg|atc|tac|- <4>|<4>|<4>|<2>|cgt|cct|tct|ggt|gtc|agc|aat|c-3′ CDR3 (Lm3TSh822): 5′-ctg|cag|gct|gaa|gac|gag|gct|gac-3′ (Lm3TLo819): 5′-ctg|cag|gct|gaa|gac|gag|gct|gac|tac|tat|tgt|-3′ (Lm3BLo825) [RC]: 5′-gtc|ttc|ggc|ggt|ggt|acc|aaa|ctt|act|gtc|ctc|ggt|caa|cct|aag|g- acacaggtgag-3′ (Lm3BSh832) [RC]: 5′-c|ggt|caa|cct|aag|gacacaggtgag-3′ (Lm3vg817): 5′-gac|gag|gct|gac|tac|tat|tgt|- |<4>|<5>|<4>|<2>|<4>|tct|<4>|<4>|<4>|<4>|-             Gtc|ttc|ggc|ggt|ggt|acc|aaa|ctt|ac-3′ (Lm3vg817-10): 5′-gac|gag|gct|gac|tac|tat|tgt|- |<5>|agc|tat|<1>|<5>|tct|<5>|<1>|<4>|gtc|ttc|ggc|ggt|ggt|- |acc|aaa|ctt|ac-3′

TABLE 10 A27:JH1 Kappa light chain gene with stuffers in place of CDRs Each stuffer contains at least one stop codon and a restriction site that will be unique within the diversity vector. gaggacc attgggcccc ctccgagact ctcgagcgca   Scab.....EcoO109I            ApaI.                               XhoI.. acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc       ..-35..         Plac                    ..-10. cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatgac catgatta cgccaagctt tggagccttt tttttggaga ttttcaac           PflMI.......             Hind3. M13 III signal sequence (AA seq)--------------------------->  1   2   3   4   5   6   7   8   9  10  11  12  13  14  15  M   K   K   L   L   F   A   I   P   L   V   V   P   F   Y gtg aag aag ctc cta ttt gct atc ccg ctt gtc gtt ccg ttt tac --Signal-->  FR1------------------------------------------->  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30   S   H   S   A   Q   S   V   L   T   Q   S   P   G   T   L |agc|cat|agt|gca|caa|tcc|gtc|ctt|act|caa|tct|cct|ggc|act|ctt|           ApaLI... ----- FR1 --------------------------------->|-------Stuffer->  31  32  33  34  35  36  37  38  39  40  41  42  43   S   L   S   P   G   E   R   A   T   L   S   |   | |tcg|cta|agc|ccg|ggt|gaa|cgt|gct|acc|tta|agt|tag|taa|gct|ccc|    EspI.....                       AflII...            XmaI.... - Stuffer for CDR1--> FR2 ------- FR2 ------>|-----------Stuffer for CDR2                        59  60  61  62  63  64  65  66                         K   P   G   Q   A   P   R |agg|cct|ctt|tga|tct|g|aaa|cct|ggt|cag|gcg|ccg|cgt|taa|tga|aagcgctaatggccaacagtg  StuI...                 SexAI...    KasI....              AfeI..   MscI.. Stuffer-->|--- FR3 ----------------------------------------------->  76  77  78  79  80  81  82  83  84  85  86  87  88  89  90   T   G   I   P   D   R   F   S   G   S   G   S   G   T   D |act|ggg|atc|ccg|gac|cgt|ttc|tct|ggc|tct|ggt|tca|ggt|act|gac|       BamHI...              RsrII..... ------ FR3 ----->----------------STUFFER for CDR3------------------>  91  92  93  94  95  96  97   F   T   L   T   I   S   R   |   | |ttt|acc|ctt|act|att|tct|aga|taa|tga| gttaac tag acc tacgta acc tag                      XbaI...          HpaI..         SnaBI. -----------------CDR3 stuffer------------------>|-----FR4--->                                                 118 119 120                                                   F   G   Q                                                 |ttc|ggt|caa| -----FR4------------------->|      <------- Ckappa ------------ 121 122 123 124 125 126 127        128 129 130 131 132 133 134   G   T   K      V   E   I   K     R   T   V   A   A   P   S |ggt|acc|aag|gtt|gaa|atc|aag| |cgt|acg|gtt|gcc|gct|cct|agt|       StyI....                 BsiWI..  135 136 137 138 139 140 141 142 143 144 145 146 147 148 149   V   F   I   F   P   P   S   D   E   Q   L   K   S   G   T |gtg|ttt|atc|ttt|cct|cct|tct|gac|gaa|caa|ttg|aag|tca|ggt|act|                                      MfeI... acgcatctctaa gcggccgc aacaggaggag             NotI....              EagI..

TABLE 11 2a2:JH2 Human lambda-chain gene with stuffers in place of CDRs gaggaccatt gggcccc   ttactccgtgac Scab...... EcoO109I            ApaI..         -----------FR1-------------------------------------------->          1   2   3   4   5   6   7   8   9  10  11  12  13  14  15  S   A   Q   S   A   L   T   Q   P   A   S   V   S   G   S   P   G agt|gca|caa|tcc|gct|ctc|act|cag|cct|gct|agc|gtt|tcc|ggg|tca|cct|ggt|  ApaLI...                           NheI...          BstEII...                                                           SexAI.... ------FR1------------------> |-----stuffer for CDR1---------  16  17  18  19  20  21  22  23   Q   S   I   T   I   S   C   T |caa|agt|atc|act|att|tct|tgt|aca|tct tag tga ctc                          BsrGI.. -----Stuffer--------------------------->-------FR2----------> 31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  R   S   |   |   P   |                     H   P   G   K   A aga tct taa tga ccg tag                 cac|ccg|ggc|aag|gcg|  BglII                                     XmaI....    KasI.....                                            AvaI.... --|-------------Stuffer for CDR2 ------------------------------------->  P |ccg|taa|tga|atc tcg tac g                               ct|ggt|gtt| KasI....          BsiWI... -------FR3----------------------------------------------------  61  62  63  64  65  66  67  68  69  70  71  72  73  74  75   S   N   R   F   S   G   S   K   S   G   N   T   A   S   L |agc|aat|cgt|ttc|tcc|gga|tct|aaa|tcc|ggt|aat|acc|gca|agc|tta|                  BspEI..                           HindIII.                       BsaBI........(blunt) -------FR3------------->|--Stuffer for CDR3----------------->|  76  77  78  79  80  81  82  83  84  85  86  87  88  89  90   T   I   S   G   L   Q |act|atc|tct|ggt|ctg|cag|gtt ctg tag ttc caattg ctt tag tga ccc                  PstI...                 MfeI.. -----Stuffer------------------------------->|---FR4---------                                                 103 104 105                                                  G   G   G                                                |ggc|ggt|ggt|                                                          KpnI... -------FR4-------------->  106 107 108 109 110 111 112 113 114 115 116 117 118 119 120   T   K   L   T   V   L   G   Q   P   K   A   A   P   S   V |acc|aaa|ctt|act|gtc|ctc|ggt|caa|cct|aag|gct|gct|cct|tcc|gtt| KpnI...                   HincII..                                  Bsu36I...  121 122 123 124 125 126 127 128 129 130 131 132 133 134 135   T   L   F   P   P   S   S   E   E   L   Q   A   N   K   A |act|ctc|ttc|cct|cct|agt|tct|gaa|gag|ctt|caa|gct|aac|aag|gct|                              SapI.....  136 137 138 139 140 141 142 143 144 145 146 147 148 149 150   T   L   V   C   L   I   S   D   F   Y   P   G   A   V   T |act|ctt|gtt|tgc|ttg|atc|agt|gac|ttt|tat|cct|ggt|gct|gtt|act|                   BclI....

The invention relates to generation of useful diversity in synthetic antibody (Ab) gene, especially to Ab genes having frameworks derived from human Abs.

BACKGROUND OF THE INVENTION

Antibodies are highly useful molecules because of their ability to bind almost any substance with high specificity and affinity and their ability to remain in circulation in blood for prolonged periods as therapeutic or diagnostic agents. For treatment of humans, Abs derived from human Abs are much preferred to avoid immune response to the Ab. For example, murine Abs very often cause Human Anti Mouse Antibodies (HAMA) which at a minimum prevent the therapeutic effects of the murine Ab. For many medical applications, monoclonal Abs are preferred. Nowadays the preferred method of obtaining a human Ab having a particular binding specificity is to select the Ab from a library of human-derived Abs displayed on a genetic package, such as filamentous phage.

Libraries of phage-displayed Fabs and scFvs have been produced in several ways. One method is to capture the diversity of donors, either naive or immunized. Another way is to generate libraries having synthetic diversity. The present invention relates to methods of generating useful diversity in human Ab scaffolds.

As is well known, typical Abs consist of two heavy chains (HC) and two light chains (LC). There are several types of HCs: gamma, mu, epsilon, delta, etc. Each type has an N-terminal V domain followed by three or more constant domains. The LCs comprise an N-terminal V domain followed by a constant domain. LCs come in two types: kappa and lambda.

Within each V domain (LC or HC) there are seven canonical regions, named FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, where “FR” stands for “Framework Region” and “CDR” stands for “Complementarity Determining Region”. For LC and HC, the FR and CDR GLGs have been selected over time to be secretable, stable, non-antigenic and these properties should be preserved as much as possible. Actual Ab genes contain mutations in the FR regions and some of these mutations contribute to binding, but such useful FR mutations are rare and are not necessary to obtain high-affinity binding. Thus, the present invention will concentrate diversity in the CDR regions.

In LC, FR1 up to FR3 and part of CDR3 comes from a genomic collection of genes called “V-genes”. The remainder of CDR3 and FR4 comes from a genomic collection of genes called “J-genes”. The joining may involve a certain degree of mutation, allowing diversity in CDR3 that is not present in the genomic sequences. After the LC gene is formed, somatic mutations can give rise to mature, rearranged LC genes that have higher affinity for an antigen (Ag) than does any LC encoded by genomic sequences. A large fraction of somatic mutations occur in CDRs.

The HC V region is more complicated. A V gene is joined to a J gene with the possible inclusion of a D segment. About half of HC Abs sequences contain a recognizable D segment in CDR3. The joining is achieved with an amazing degree of molecular sloppiness. Roughly, the end of the V gene may have zero to several bases deleted or changed, the D segment may have zero to many bases removed or changed at either end, a number of random bases may be inserted between V and D or between D and J, and the 5′ end of J may be edited to remove or change several bases. Withal, it is amazing that human heavy chains work, but they do. The upshot is that the CDR3 is highly diverse both in encoded amino-acid sequences and in length. In designing synthetic libraries, there is the temptation to just throw in a high degree of synthetic diversity and let the phage sort it out. Nevertheless, D regions serve a function. They cause the Ab repertoire to be rich in sequences that a) allow Abs to fold correctly, and b) are conducive to binding to biological molecules, i.e. antigens.

One purpose of the present invention is to show how a manageable collection of diversified sequences can confer these advantages on synthetic Ab libraries. Another purpose of the present invention is to disclose analysis of known mature Ab sequences that lead to improved designs for diversity in the CDR1 and CDR2 of HC and the three CDRs of lambda and kappa chains.

BRIEF STATEMENT OF THE INVENTION

The invention is directed to methods of preparing synthetically diverse populations of Ab genes suitable for display on genetic packages (such as phage or phagemids) or for other regimens that allow selection of specific binding. Said populations concentrate the diversity into regions of the Ab that are likely to be involved in determining affinity and specificity of the Ab for particular targets. In particular, a collection of actual Ab genes has been analyzed and the sites of actual diversity have been identified. In addition, structural considerations were used to determine whether the diversity is likely to greatly influence the binding activity of the Ab. Schemes of variegation are presented that encode populations in which the majority of members will fold correctly and in which there is likely to be a plurality of members that will bind to any given Ag. Specifically, a plan of variegation is presented for each CDR of the human heavy chain, kappa light chain, and lambda light chain. The variegated CDRs are presented in synthetic HC and LC frameworks.

In one embodiment, the invention involves variegation of human HC variable domains based on a synthetic 3-23 domain joined to a JH4 segment in which the variability in CDR1 and CDR2 comprises sequence variation of segments of fixed length while in CDR3 there are several components such that the population has lengths roughly corresponding to lengths seen in human Abs and having embedded D segments in a portion of the longer segments. In the light chains, the kappa chain is built in an A27 framework and a J1(1 while lambda is built in a 2a2 framework with an L2 J region.

EXAMPLES Choice of a Heavy-Chain V Domain

The HC Germ-Line Gene (GLG) 3-23 (also known as VP-47) accounts for about 12% of all human Abs and it suitable for the framework of the library. Certain types of Ags elicit Abs having particular types of VH genes; in some cases, the types elicited are otherwise rarely found. This apparent Ag/Ab type specificity has been ascribed to possible structural differences between the various families of V genes. It is also possible that the selection has to do with the availability of particular AA types in the GLG CDRs. Suppose, for example, that the sequence YR at positions 4 and 5 of CDR2 is particularly effective in binding a particular type of Ag. Only the V gene 6-1 provides this combination. Most Abs specific for the Ag will come from GLG 6-1. If Y4-R5 were provided in other frameworks, then other frameworks are likely to be as effective in binding the Ag.

Analysis of HC CDR1 and CDR2:

In CDR1 and CDR2 of HCs, the GLGs provide limited length diversity as shown in Table 15P. Note that GLGs provide CDR1s only of the lengths 5, 6, and 7. Mutations during the maturation of the V-domain gene leads to CDR1s having lengths as short as 2 and as long as 16. Nevertheless, length 5 predominates. The preferred length for the present invention is 5 AAs in CDR1 with a possible supplemental components having lengths of 7 and 14.

GLGs provide CDR2s only of the lengths 15-19, but mutations during maturation result in CDR2s of length from 16 to 28 AAs. The lengths 16 and 17 predominate in mature Ab genes and length 17 is the most preferred length for the present invention. Possible supplementary components of length 16 and 19 may also be incorporated.

Table 20P shows the AA sequences of human GLG CDR1s and CDR2. Table 21P shows the frequency of each amino-acid type at each position in the GLGs. The GLGs as shown in Table 20P have been aligned by inserting gaps near the middle of the segment so that the ends align.

The 1398 mature V-domain genes used in studying D segments (vide infra) were scanned for examples in which CDR1 and CDR2 could be readily identified. Of this sample 1095 had identifiable CDR1, 2, and 3. The CDRs were identified by finding subsequences of the GLGs in an open reading frame. There are 51 human HC V genes. At the end of FR1, there are 20 different 9-mers. At the start of FR2, there are 11 different 9-mers. At the end of FR2 there are 14 different 9-mers. At the start of FR3, there are 14 different 9-mers. At the end of FR3, there are 13 different 9-mers. At the start of JH, there are three different 9-mers. These motifs were compared to the reported gene in frame and a match, at the site of maximum similarity, of seven out of nine was deemed acceptable. Only when all three CDRs were identified were any of the CDRs included in the analysis. In addition, the type of the gene was determined by comparing the framework regions to the GLG frameworks; the results are shown in Table 22P.

Design of HC CDR1 and CDR2 Diversity

Diversity in CDR1 and CDR2 was designed from: a) the diversity of the GLGs, b) observed diversity in mature HC genes, and c) structural considerations. In CDR1, examination of a 3D model of a humanized Ab showed that the side groups of residues 1, 3, and 5 were directed toward the combining pocket. Consequently, we allow each of these positions to be any amino-acid type except cysteine. Cysteine can form disulfide bonds. Disulfide bonds are an important component of the canonical Ig fold. Having free thiol groups could interfere with proper folding of the HC and could lead to problems in production or manipulation of selected Abs. Thus, I exclude cysteine from the menu. The side groups of residue 2 is directed away from the combining pocket. Although this position shows substantial diversity, both in GLG and mature genes, I fixed this residue as Tyr because it occurs in 681/820 mature genes (Table 21P). Position 4 is fixed as Met. There is some diversity here, but almost all mature genes have uncharged hydrophobic AA types: M, W, I, V, etc. (Table 21P). Inspection of a 3D model shows that the side group of residue 4 is packed into the innards of the HC. Since we are using a single framework (3-23), we retain the Met that 3-23 has because it is likely to fit very well into the framework of 3-23. Thus, the most preferred CDR1 library consists of XYXMX where X can be any one of [A,D,E,F,G,H,I,K,L,M,N,P,Q,R,S,T,V,W,Y] (no C). The DNA that encodes this is preferably synthesized using trinucleotide building blocks so that each AA type is present in essentially equimolar amounts. Specifically, the X codons are synthesized using a mixture of the codons [gct, gat, gag, ttt, ggt, cat, att, aag, atg, aat, cct, cag, cgt, tct, act, gtt, tgg, tat]. This diversity is shown in the context of a synthetic 3-23 gene in Table 18P. The diversity oligonucleotide (ON) is synthesized from BspEI to BstXI and can be incorporated either by PCR synthesis using overlapping ONs or introduced by ligation of BspEI/BstXI-cut fragments. Table 22P shows ONs that embody the specified variegation. PCR using ON-R1V1vg, ON-R1 top, and ON-R1bot gives a dsDNA product of 73 base pairs, cleavage with BspEI and BstXI trims 11 and 13 bases from the ends and provides cohesive ends that can be ligated to similarly cut vector having the synthetic 3-23 domain shown in Table 18P. Replacement of ON-R1V1vg with either ONR1V2vg or ONR1V3vg allows synthesis of the two alternative diversity patterns given below.

Alternatively, one can include CDR1 s of length 7 and/or 14. For length 7, a preferred diversity is (S/T)₁(S/G/x)₂(S/G/x)₃Y₄Y₅W₆(S/G/x)₇; where (S/T) indicates an equimolar mixture of Ser and Thr codons; (S/G/x) indicates a mixture of 0.2025 S, 0.2025 G, and 0.035 for each of A, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, Y. Other proportions could be used. The design gives a predominance of Ser and Gly at positions 2, 3, and 7, as occurs in mature HC genes. For length 14, a preferred pattern of diversity is VSGGSISXXXYYWX where X can be any AA type except Cys. This pattern appears to arise by insertions into the GLG sequences (SGGYYWS (4-30.1 and 4-31) and similar sequences. There is a preference for a hydrophobic residue at position 1 (V or C) with a second insertion of SISXXX between GG and YY. Diversity ONs having CDR1s of length 7 or 14 are synthesized from BspEl to BstXI and introduced into the library in appropriate proportions to the CDR1 of length 5. The components should be incorporated in approximately the ratios in which they are observed in antibodies selected without reference to the length of the CDRs. For example, the sample of 1095 HC genes examined here have them in the ratios (L=5:L=7:L=14::820:175:23::0.80:0.17:0.02).

CDR2

Diversity at CDR2 was designed with the same considerations: GLG sequences, mature sequences and 3D structure. A preferred length for CDR2 is 17, as shown in Table 18P. Examination of a 3D model suggests that the residues shown as varied in Table 18P are the most likely to interact directly with Ag. Thus a preferred pattern of variegation is: <2>I<2><3>SGG<1>T<1>YADSVKG, where <2> indicates a mixture of YRWVGS, <3> is a mixture of P and S, and <1> is a mixture of ADEFGHIKLMNPQRSTVWY (no C). ON-R2V1vg shown in Table 22P embodies this diversity pattern. PCR with ON-R2V1vg, ON-R2top, and ONR2bot gives a dsDNA product of 122 base pairs. Cleavage with BstXI and XbaI removes about 10 bases from each end and produces cohesive ends that can be ligated to similarly cut vector that contains the 3-23 gene shown in Table 18P.

An alternative pattern would include the variability seen in mature CDR2s as shown in Table 21P: <1>I<4><1><1>G<5><1><1><1>YADSVKG, where <4> indicates a mixture of DINSWY, and <5> indicates a mixture of SGDN. This diversity pattern is embodied in ON-R2V2vg shown in Table 22P. For either case, the variegated ONs would be synthesized so that fragments of dsDNA containing the BstXI and XbaI site can be generated by PCR. ON-R2V2vg embodies this diversity pattern.

Alternatively, one can allow shorter or longer CDR2s. Table 22P shows ON-R2V3vg which embodies a CDR2 of length 16 and ON-R2V4vg which embodies a CDR2 of length 19. Table 22P shows ON-R1V3vg is PCR amplified with ON-R2top and ON-R2bo3 while ON-R2V4vg is amplified with ON-R2top and ONR2-bo4.

Analysis of HC CDR3:

CDR3s of HC vary in length and in sequence. About half of human HCs consist of the components: V::nz::D::ny::JHn where V is a V gene, nz is a series of bases (mean 12) that are essentially random, D is a D segment, often with heavy editing at both ends, ny is a series of bases (mean 6) that are essentially random, and JH is one of the six JH segments, often with heavy editing at the 5′ end. In HCs that have no identifiable D segment, the structure is V::nz::JHn where JH is usually edited at the 5′ end. Our goal is to mimic the diversity of CDR3, but not to duplicate it (which would be impossible). The D segments appear to provide spacer segments that allow folding of the IgG. The greatest diversity is at the junctions of V with D and of D with JH. The planned CDR3 library will consist of several components. Some of these will have only sequence diversity. Others will have sequence diversity with embedded D segments to extend the length while incorporating sequences known to allow Igs to fold.

There are many papers on D segments. Corbett et al. (1997) show which D segments are used in which reading frames. My analysis basically confirms their findings. They did not report, however, the level of editing of each D segment and this information is needed for design of an effective library.

The following diversified sequences would be incorporated in the indicated proportions: “I” stands for 0.095 [G, Y] and 0.048 [A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W]; double dose of Gly and Tyr plus all other AAs except Cys at equal level.

The amount of each component is assigned from the tabulation of lengths of the collection of natural NTH genes. Component 1 represents all the genes having length 0 to 8 (counting from the YYCAR motif to the WG dipeptide motif). Component 2 corresponds the all the chains having length 9 or 10. Component 3 corresponds to the genes having length 11 or 12 plus half the genes having length 13. Component 4 corresponds to those having length 14 plus half those having length 13. Component 5 corresponds to the genes having length 15 and half of those having length 16. Component 6 corresponds to genes of length 17 plus half of those with length 16. Component 7 corresponds to those with length 18. Component 8 corresponds to those having length 19 and greater.

The composition has been adjusted because the first component is not complex enough to justify including it as 10% of the library. If the final library were to be 1. E 9, then 1. E 8 sequences would come from component 1, but it has only 2.6 E 5 CDR3 sequences so that each one would occur in ˜385 CDR1/2 contexts. I think it better to have this short CDR3 diversity occur in ˜77 CDR1/2 contexts and have the other, longer CDR3s occur more often.

The ONs would be PCR amplified with the primers CtprmA and CBprmB, cut with AflII and BstEH, and ligated to similarly cut V3-23.

This set of components was designed after studying the sequences of 1383 human HC sequences as described below. The proposed components are meant to fulfill the goals:

1) approximately the same distribution of lengths as seen in real Ab genes, 2) high level of sequence diversity at places having high diversity in real Ab genes, and 3) incorporation of constant sequences often seen in real Ab genes.

Note that the design uses JH4 (YFDYWGQGTLVTVSS), which is found more often, instead of JH3 (AFDIWGQGTMVTVSS). This involves three changes in AA sequence, shown as double underscored bold. An alternative JH segment is shown.

How the Library Components were Designed:

The processing of sequence data was accomplished by a series of custom-written FORTRAN programs, each of which carries out a fairly simple transformation on the data and writes its results as one or more ASCII files. The next program then uses these files as input.

A set of 2049 human heavy-chain genes was selected from the version of GenBank that was available at Dyax on the Sun server on 26 Jun. 2000. A program named “Reformat” changed the format of the files to that of GenBank from the GCG format, creating one file per sequence. A second program named “IDENT_CDR3” processed each of these files as follows. Files were tested for duplication by previous entries, duplicates were discarded. Each reading frame was tested. Most entries had a single open reading frame (ORF), none had two, and some had none. Entries with multiple stops in every reading frame were discarded because this indicates poor quality of sequencing. The sequence was written in triplets in the ORF or in all three reading frames if no ORF was found. The sequence was examined for three motifs: a) AA sequence=“YYCxx”, b) DNA sequence=“tgg ggc (=WG)”, and DNA sequence=“g gtc acc (=BstEII)”. FR3 ends with a conserved motif YYCAR or a close approximation. When writing the DNA sequence, IDENT_CDR3 prints the DNA mostly in lower case. Cysteine codons (TGT or TGC) are printed in uppercase. When the motif “tay tay tgy” is found, IDENT_CDR3 starts a new line that contains “< > xxx xxx xxx xxx xxx” where the xxx's stand for the actual five codons that encode YYC and the next two codons (most often AR or AK). The following DNA is printed in triplets on new lines. A typical processed entry appears as in Table 1P.

Following the YYC motif, IDENT_CDR3 seeks the sequence “TGG GGC” (the “WG” motif) in the correct reading frame, 5/6 bases is counted as a hit. If found, the DNA is made uppercase. Following the WG motif (if found) or the YYC motif (if no WG found), IDENT_CDR3 seeks the sequence “G GTC ACC” (the BstEII site) in the correct reading frame, 6/7 bases is counted as a hit. If found, the bases are made upper case. If either the WG or BstEII motif are not found, a note is inserted saying that the feature was not identified. The output of IDENT_CDR3 was processed by hand. In many cases, the lacking YYC motif could be seen as a closely related sequence, such as YFC, FYC, or HYC. When this was supported by an appropriately positioned WG and/or BstEII site, the effective YYC site was marked and the sequence retained for further analysis. If the YYC motif could not be identified or if the WG or BstEII sites could not be found, the entry was discarded. For example, the entry in Table 2P had no YYC motif.

The double underscored sequence encodes YHCAS and is taken as the end of FR3. Note that there is a WG motif at bases 403-408 (bold upper case) and a BstEII site at bases 420-426 (bold upper case). Using WordPerfect, I first made all occurrences of TGC and TGT bold. I then searched for “YYC not found”. If I could see the “YYC”-related sequence quickly, I edited the entry so that a YYC was shown. The entry above would be converted to that shown in Table 3P. This processing reduced the list of entries to 1669.

A third program named “New_DJ” processed the output of IDENT_CDR3. The end of the YYC motif (including the two codon following TGy=Cys) was taken as the end of FR3. The WG motif was taken as the end of the region that might contain a D segment. If WG was not observed and BstEII was, the WG site was assumed to be 17 bases upstream of BstEII. Using the WG motif for alignment, the sequence was compared to each human GLG JH segment (1-6) and the best one identified (New_DJ always assigned a JH segment). Starting from the WG motif of JH and moving toward the 5′ end, the program looked for the first codon having more than one mismatch. The region from YYCxx to this codon was taken as the region that might contain a D segment.

The region that might contain a D segment was tested against all the germ-line genes (GLGs) of human D segments and the best D segment was identified. The scoring involved matching the observed sequence to the GLG sequence in all possible ways. Starting at each base, multiply by 4 for a match and divide by 4 for a mismatch. Record the maximum value obtained for this function. The match was deemed significant if 7/7, 8/9, 9/11, etc. or more bases matched. Of the 1383 sequences examined for D segments,

“Assign_D” processes the output of New_DJ. For each sequence that had a significant match with a GLG D segment, a file was written containing the putative D segment, the DJ segment, the identified GLG D segment, the identified JH segment, the phase of the match between observed and GLG gene. For example, “D1_(—)1-01_Phz0_hsa239356.txt” is a file recording the match of entry hsa239356 with D1-01 in phase 0. The file contains the information shown in Table 4P. The final DV of the second sequence immediately precedes the WG in JH and is ascribed to JH3. Other files that begin D1_(—)1-01_Phz0 match the same GLG D segment and these can be aligned by sliding amino-acid sequences across each other.

Table 5P shows how sequence hs6d4xb7 is first assigned to JH4 and then to D3-22. Note that the DNA sequence TGGGGG is aligned to the TGG GGC of the GLG and that the sequence is truncated on the left to fit. The program finds that JH4 has the best fit (5 misses and 18 correct out of 23). From the right, the program sees that DYWGQ (underscored) come from JH, but then the match drops off and the rest of the sequence on the left comes either from added bases or a D segment.

The lower part of Table 5P shows that the possible D segment matches D#13 (3-23) is a very good match.

Of 1383 files accepted by Assign_D, 757 had identifiable D segments. The tally of JHs in Table 6P shows that JH4 is by far the most common.

JH4 is most common, JH6 next, followed by JH3 and JH5. JH1 and JH2 are seldom used. Table 7P shows the length distributions of each JH class; they do not differ significantly class to class. These lengths count only amino-acids that are not accounted for by JH and so are shorter that the lengths given in Table 8P which cover from YYCAR to WG.

Table 8P contains the distribution of lengths for a) all the CDR3 segments, b) the CDR3 segments with identified D segments, and c) the CDR3 segments having no identifiable D segment. The CDR3s with identifiable D segments (13.9) are systematically longer than are those that lack D segments (11.2).

The identified CDR3 segments can be collated in two ways: aligned to the left (looking for a pattern following YYCAR) or aligned to the right (looking for a pattern preceding WG). Table 9P shows the collation of left-aligned sequences while Table 10P shows the right-aligned sequences. For each position, I have tabulated the frequency of each AA type (A-M in the first block and N-Y in the second). The column headed “#” shows how many sequences have some AA at that position. The final column shows all of the AA types seen at that position with the most frequent first and the least frequent last. In the left-aligned sequences, we see that Gly is highly over-represented in the first seven positions while Tyr is over-represented at positions 8-16.

In Table 11P, I have tabulated the AA frequencies for the sequences having between 7 and 15 AAs between YYCAR and WG. The last four positions can be viewed as coming from JH and so would be given lower levels of diversity than would earlier positions. From these tabulations, I conclude that most AA types are allowed at all the positions, but there is a fairly strong tendency to have Gly at the early positions and to end in Asp-Tyr (DY). We could use these tendencies in designing a pattern of variegation. I would not exclude any AA except Cys, but I might increase the frequency of Gly in the first several positions and Tyr in the last few.

There are 80 sequences (5.8%) having a pair of cysteines in CDR3. It is more surprising that 53 (3.8%) have a single Cys in CDR3.

MS-DOS was used to make a list of the files written by Assign_D. “Filter” converts the output of MS-DOS Dir into a form that can be read into WordPerfect and sorted to bring a files belonging to the same D region together.

“Filter2” collects the sequences and produces a draft table of sequences, grouped by the D-segment used, and written so that the sequences can be aligned. The output of Filter2 were edited by hand. For each group, the translation of the GLG was inserted and the collection of observed sequences was aligned to the conserved part of the GLG. “Filter3” collated the aligned sequences. Table 12P shows an example of an alignment and the tabulation of AA types. The entries are as follows: “Entry” is the name used in the data base, “Seq1” is the sequence from the YYCAR motif to the first amino acid not assigned to JH and “L1” is the length of the segment. The segments are shown aligned to the identified D segment. Seq2 is the sequence from the YYCAR motif to the WG motif (i.e. including part of JH) and “L2” is the length of that sequence. JH is the identified JH segment for this sequence. “P” is the phase of the match. For positive values of P, P bases are found in the observed sequence that do not correspond to any from the GLG, i.e. the observed sequence has had that many bases inserted. For negative values of P, there are bases in the GLG sequence for which there are no corresponding bases in the observed sequence. “Score” is approximately 1/(probability of accidental match). This is calculated by looking at all possible alignments. For each alignment, the score is first set to 1.0. Base by base, the score is multiplied by 4. if the bases match and divided by 4. if they do not. This is done for all starting points and ending points and the maximum value is recorded.

Table 13P is a summary of how often each D segment was identified and in which reading frame. I have not been consistent with Corbett et al. in assigning the phases of the GLG D segments. The MRC Web page that I took the GLGs from did not have D segments D1-14, D4-11, D5-18, or D6-25. None of these contribute to any great extent and this omission is unlikely to have any serious effect on the conclusions. The column headed “%” contains the percentage of the sequences examined here. The column headed “C %” contains the percentage reported by Corbett et al. I assume that the data used in Corbett et al. is mostly included in my collection. Nevertheless, the observed frequencies differ in detail. For example, my compilation shows that 10.7% of the collection contains a D segment encoding two cysteines while they have only 4.16% in this category. In D3 phase “0”, I see 19.4% of the collection while they report 11.8%.

The most common actual D segments were further analyzed. The GLGs are heavily edited at either end. The aligned sequences were aligned. For each D-segment having more than seven examples, Filter3 produced a table of the frequency of each amino-acid type at each position. From these tabulations, library components shown in Table 17P were designed. At each position where at least half the examples have an amino acid, I entered either the dominant AA type or “x”. An AA type was “dominant” if it occurred more than 50% of the time. L is the length and f is the number of sequences observed that have related sequences.

Table 14P shows possible library components for a library of CDR3's. “L” is the length of the insert and “f” is the frequency of the motif in the assayed collection. Table 17P shows vgDNA that embodies each of the components shown in Table 14P. In Table 17P, the oligonucleotides (ON) Ctop25, CtprmA, CBprmB, and CBot25 allow PCR amplification of each of the variegated ONs (vgDNA): C1t08, C2t10, C3t12, C4t14, C5t15, C6t17, C7t18, and c8t19. After amplification, the dsDNA can be cleaved with AflII and BstEII (or Kpnl) and ligated to similarly cleaved vector that contains the remainder of the 3-23 synthetic domain. Preferably, this vector already contains diversity in CDR1 and CDR2 as disclosed herein. Preferably, the recipient vector contains a stuffer in place of CDR3 so that there will be no parental sequence that would then occur in the resulting library. Table 50P shows a version of the V3-23 gene segment with each CDR replaced by a short segment that contains both stop codons and restriction sites that will allow specific cleavage of any vector that does not have the stuffer removed. The stuffer can either be short and contain a restriction enzyme site that will not occur in the finish library, allowing removal of vectors that are not cleaved by both AfIII and BstEII (or KpnI) and religated. Alternatively, the stuffer could be 200-400 bases long so that uncleaved or once cleaved vector can be readily separated from doubly cleaved vector.

In the vgDNA for HC CDR3, <1> means a mixture comprising 0.27 Y, 0.27 G, and 0.027 of each of the amino-acid codons {A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W}; <2> means an equimolar mixture of K and R; and <3> means an equimolar mixture of S and G.

Analysis of Human Kappa Light Chains and Preferred Variegation Scheme:

A collection of 285 human kappa chains was assembled from the public data base. Table 27 shows the names of the entries used. The GLG sequences of nine bases at each end of the framework regions were used to find the FR/CDR junctions. Only in cases where all six junctions could be found was the sequences included. Table 25P shows the distribution of lengths in CDRs in human kappas. CDR1s with lengths of 11, 12, 13, 16, and 17 were observed with 11 being predominant and 12 well represented. CDR2 exhibits only length 7. CDR3 exhibits lengths of 1, 4, 6, 7, 8, 9, 10, 11, 12, 13, and 19. Essentially all examples are in the 8, 9, or 10 length groups.

Table 26P shows the distribution of V and J genes seen in the sample. A27 is the most common V and JK1 is the most common J. Thus, a suitable synthetic kappa gene comprises A27 joined to JK1. Table 30P shows a suitable synthetic kappa chain gene, including a PlacZ promoter, ribosome-binding site, and signal sequence (M13 III signal). The DNA sequence encodes the GLG amino-acid sequence, but does not comprise the GLG DNA sequence. Restriction sites are designed to fall within each framework region so that diversity can be cloned into the CDRs. XmaI and EspI are in FR1, SexAI is in FR2, RsrII is in FR3, and Kpnl (or Acc65I) are in FR4. Additional sites are provided in the constant kappa chain to facilitate construction of the gene.

Table 30P also shows a suitable scheme of variegation for kappa. In CDR1, a preferred length is 11 codons. The A27 GLG has a CDR1 of 12 codons, but the sample of mature kappa chains has length 11 predominating. One could also introduce a component of kappas having length 12 in CDR1 by introducing codon 52 as <2> (i.e. a Ser-biased mixture). CDR2 of kappa is always 7 codons. Table 31P shows a tally of 285 CDR2s and a preferred variegation scheme for CDR2. The predominant length of CDR3 in kappa chains is 9 codons. Table 32P shows a tally of 166 CDR3s from human kappas and a preferred variegation scheme (which is also shown in Table 30P).

Analysis of Lambda Chains and Preferred Variegation Scheme:

A collection of 158 lambda sequences was obtained from the public data base. Of these 93 contained sequences in which the FR/CDR boundaries could be identified automatically. Table 33P shows the distribution of lengths of CDRs.

Method of Construction:

The diversity of HC, kappa, and lambda are best constructed in separate vectors. First a synthetic gene is designed to embody each of the synthetic variable domains. The light chains are bounded by restriction sites for ApaLI (positioned at the very end of the signal sequence) and AscI (positioned after the stop codon). The heavy chain is bounded by SfiI (positioned within the PelB signal sequence) and NotI (positioned in the linker between CH1 and the anchor protein. The initial genes are made with “stuffer” sequences in place of the desired CDRs. A “Stuffer” is a sequence the is to be cut away and replaced by diverse DNA but which does not allow expression of a functional antibody gene. For example, the stuffer may contain several stop codons and restriction sites that will not occur in the correct finished library vector. In Table 40P, the stuffer for CDR1 of kappa A27 contains a StuI site. The vgDNA for CDR1 is introduced as a cassette from EspI, XmaI, or VII to either SexAI or KasI. After the ligation, the DNA is cleaved with StuI; there should be no StuI sites in the desired vectors.

REFERENCES

-   Corbett, S J, Tomlinson, I M, Sonnhammer, E L L, Buck, D, Winter, G.     “Sequences of the Human Immunoglobulin Diversity (D) Segment Locus:     A Systematic Analysis Provides No Evidence for the Use of DIR     Segments, Inverted D Segments, ‘Minor’ D Segments or D-D     Recombination”. J Molec Biol (1997) 270:587-597.

TABLES

TABLE 1P Typical entry in which YYC motif is found. ++++C:\tmp\haj10335.txt LOCUS HAJ10335      306 bp    mRNA          PRI        18-AUG-1998 DEFINITION Homo sapiens mRNA for immunoglobulin heavy chain variable region, clone ELD16/6. ACCESSION AJ010335 VERSION AJ010335.1 GI: 3445266   Ngene = 306  Stop codons in reading frame 1     49 115 124 253 277  No stops in reading frame 2  Stop codons in reading frame 3     12 60 81 147 204 213   1   t ttg ggg tcc ctg aga ctc tcc TGT gca gcc tct gga ttc acc  44 gtc agt agc aac tac atg acc tgg gtc cgc cag gct cta ggg aag  89 ggg ctg gag tgg gtc tca gtt att tat agc ggt ggt agc aca tac 134 tac gca gac tcc gtg aag ggc gga ttc acc atc tcc aga gac aat 179 tcc aag aac aca ctg tat ctt caa atg aac agc ctg aga ccc gag 224 gac acg gct gtg <     > TAT TAC TGT gcg aca 251 ggt aat cgc ctg gaa atg gct gca att aac TGG GGC caa gga acc 263 ctG GTC ACC aa

TABLE 2P entry in which YYC motif was not automatically identified ++C:\tmp\hs202g3.txt !!NA_SEQUENCE 1.0 LOCUS HS202G3   522 bp  mRNA     PRI    03-AUG-1995 DEFINITION H. sapiens mRNA for immunoglobulin variable region (clone 202-G3). ACCESSION Z47259 VERSION Z47259.1 GI: 619470   Ngene = 522  No stops in reading frame 1  Stop codons in reading frame 2     89 110 305 314  Stop codons in reading frame 3     84 192 321 351 369   1 atg gac tgg acc tgg agg ttc ctc ttt gtg gtg gca gca gct aca  46 ggt gtc cag tcc cag gtg cag ctg gtg cag tct ggg gct gag gtg  91 aag aag cct ggg tcc tcg gtg aag gtc tcc TGC aag gct tct gga 136 ggc acc ttc agc agc tat gct atc agc tgg gtg cga cag gcc cct 181 gga caa ggg ctt gag tgg atg gga ggg atc atc cct atc ttt ggt 226 aca gca aac tac gca cag aag ttc cag ggc aga gtc acg att acc 271 gcg gac gaa tcc acg agc aca gcc tac atg gag ctg agc agc ctg 316 aga tct gag gac acg gcc gtg tat cac TGT gcg agt gag gga tgg 361 gag agt TGT agt ggt ggt ggc TGC tac gac ggt atg gac gtc TGG 406 GGC caa ggg acc acG GTC ACC gtc tcc tca gct tcc acc aag ggc 451 cca tcg gtc ttc ccc ctg gcg ccc TGC tcc agg agc acc tct ggg 496 ggc aca gcg gcc ctg ggc TGC ctg YYC not found !!!

TABLE 3P Entry of Table 2P after editting. ++C:\tmp\hs202g3.txt !!NA_SEQUENCE 1.0 LOCUS HS202G3 522 bp mRNA PRI 03-AUG-1995 DEFINITION H. sapiens mRNA for immunoglobulin variable region (clone 202-G3). ACCESSION Z47259 VERSION Z47259.1 GI: 619470 Ngene = 522 No stops in reading frame 1 Stop codons in reading frame 2 89 110 305 314 Stop codons in reading frame 3 84 192 321 351 369   1 atg gac tgg acc tgg agg ttc ctc ttt gtg gtg gca gca gct aca  46 ggt gtc cag tcc cag gtg cag ctg gtg cag tct ggg gct gag gtg  91 aag aag cct ggg tcc tcg gtg aag gtc tcc TGC aag gct tct gga 136 ggc acc ttc agc agc tat gct atc agc tgg gtg cga cag gcc cct 181 gga caa ggg ctt gag tgg atg gga ggg atc atc cct atc ttt ggt 226 aca gca aac tac gca cag aag ttc cag ggc aga gtc acg att acc 271 gcg gac gaa tcc acg agc aca gcc tac atg gag ctg agc agc ctg 316 aga tct gag gac acg gcc gtg <YHCAS> tat cac TGT gcg agt gag gga tgg 361 gag agt TGT agt ggt ggt ggc TGC tac gac ggt atg gac gtc TGG 406 GGC caa ggg acc acG GTC ACC gtc tcc tca gct tcc acc aag ggc 451 cca tcg gtc ttc ccc ctg gcg ccc TGC tcc agg agc acc tct ggg 496 ggc aca gcg gcc ctg ggc TGC ctg YYC not found !!!

TABLE 4P contents of file D1_1-01_Phz0_hsa239356.txt DRGGKYQLAPKGGM DRGGKYQLAPKGGMDV JH3 D# 1 Phase 15 Score 6.55D+04

TABLE 5P alignment of a CDR3::JH segment to GLG JHs and D-segments. +c:\tmp\hs6d4xb7.txt          1    1    2    2    3    3   3 1234567890    5    0    5    0    5   9 Observed tatgatagtagtgggtcatactccgactacTGGGGGcag JH1 ------------gctgaatacttccagcactggggccagggcaccctggtcaccgtctcctcag-- Miss =  9 Nt = 27 JH2 -----------ctactggtacttcgatctctggggccgtggcaccctggtcactgtctcctcag-- Miss = 13 Nt = 28 JH3 --------------tgatgcttttgatatctggggccaagggacaatggtcaccgtctcttcag-- Miss = 14 Nt = 25 JH4 ----------------actactttgactactggggccagggaaccctggtcaccgtctcctcag-- Miss =  5 Nt = 23 JH5 -------------acaactggttcgacccctggggccagggaaccctggtcaccgtctcctcag-- Miss = 11 Nt = 26 JH6 -attactactactactacggtatggacgtctggggccaagggaccacggtcaccgtctcctcag-- Miss = 23 Nt = 38   4 tat gat agt agt ggg tca TAC Tcc GAC TAC TGG GGg CAG  Y   D   S   S   G   S   Y   S   D   Y   W   G   Q JH4 --- --- --- --- --- -ac tac ttt gac tac tgg ggc cag gga acc ctg gtc acc gtc tcc tca g--  -   -   -   -   -   -   Y   F   D   Y   W   G   Q   G   T   L   V   T   V   S   S   - Fract = 0.783 = 18/23 Matching the rest to D segments: D#13 --------gtattactatgatagtagtggttattactac  GLG gatcgccacaattactatgatagtagtgggtcatactcc  Observed --------gt...................t.at....a.  . = match D#13 Phase = 9 Score = 4.3980E+12

TABLE 6P Number of sequences identified as having JH derived from GLG JHn JH 1 2 3 4 5 6 # sequences 17 40 198 707 160 261

TABLE 7P Distribution of CDR3 fragments that might contain D segments. For JH1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 0 0 1 1 3 1 1 2 0 3 1 1 1 2 Total = 17 Median = 8.0 For JH2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 0 0 0 0 2 4 6 2 6 3 4 5 2 3 15 16 17 18 2 0 0 1 Total = 40 Median = 9.0 For JH3 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 0 2 6 16 12 17 17 15 22 20 20 18 13 4 15 16 17 18 19 8 3 2 1 2 Total = 198 Median = 8.6 For JH4 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 0 7 15 19 40 63 82 81 77 81 53 57 44 30 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 15 23 8 3 5 2 0 1 0 0 0 0 0 0 0 30 31 32 33 34 35 0 0 0 0 0 1 Total = 707 Median = 8.6 For JH5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 0 0 3 4 6 13 19 12 14 22 18 10 18 10 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 5 1 1 0 0 1 1 0 0 0 0 0 0 0 0 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 45 46 0 1 Total = 160 Median = 9.4 For JH6 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 2 0 1 2 5 15 20 18 22 29 29 28 23 16 10 15 16 17 18 19 20 14 9 9 4 2 3 Total = 261 Median = 9.6

TABLE 8P Lengths of CDR3 segments from YYCAR to WG. Distribution of lengths from end of FR3 to WG motif all sequences. L 0 1 2 3 4 5 6 7 8 9 N 6 0 0 4 2 9 13 38 61 88 Sum(N) 6 6 6 10 12 21 34 72 133 221 f .004 .004 .004 .007 .009 .015 .025 .052 .096 .160 L 10 11 12 13 14 15 16 17 18 19 N 101 118 154 150 118 125 105 84 61 46 SN 322 440 594 744 862 987 1092 1176 1237 1283 f .233 .318 .430 .538 .623 .714 .790 .850 .894 .928 L 20 21 22 23 24 25 26 27 28 29 N 42 16 17 7 9 2 1 0 2 1 SN 1325 1341 1358 1365 1374 1376 1377 1377 1379 1380 f .958 .970 .982 .987 .993 .995 .996 .996 .997 .998 L 30 31 32 33 34 35 36 37 38 39 N 0 0 0 0 0 0 0 1 0 0 SN 1380 1380 1380 1380 1380 1380 1380 1381 1381 1381 f .998 .998 .998 .998 .998 .998 .998 .999 .999 .999 L 40 41 42 43 44 45 46 N 0 0 1 0 0 0 1 SN 1381 1381 1382 1382 1382 1382 1383 f .999 .999 .999 .999 .999 .999 1.0 Median = 12.65 Distribution of lengths from end of FR3 to WG motif with assigned D. L 0 1 2 3 4 5 6 7 8 9 N 3 0 0 0 0 0 3 9 21 15 SN 3 3 3 3 3 3 6 15 36 51 f .004 .004 .004 .004 .004 .004 .008 .019 .046 .065 L 10 11 12 13 14 15 16 17 18 19 N 39 64 77 97 72 77 75 63 45 35 SN 90 154 231 328 400 477 552 615 660 695 f .115 .196 .294 .418 .510 .608 .703 .783 .841 .885 L 20 21 22 23 24 25 26 27 28 29 N 38 15 15 6 9 2 1 0 1 1 SN 733 748 763 769 778 780 781 781 782 783 f .934 .953 .972 .980 .991 .994 .995 .995 .996 .997 L 30 31 32 33 34 35 36 37 38 39 N 0 0 0 0 0 0 0 1 0 0 SN 783 783 783 783 783 783 783 784 784 784 f .997 .997 .997 .997 .997 .997 .997 .999 .999 .999 L 40 41 42 43 44 45 46 N 0 0 0 0 0 0 1 SN 784 784 784 784 784 784 785 f .999 .999 .999 .999 .999 .999 1.0 Median = 13.90 Distribution of lengths from end of FR3 to WG motif with no assigned D. L 0 1 2 3 4 5 6 7 8 9 N 3 0 0 4 2 9 10 29 40 73 SN 3 3 3 7 9 18 28 57 97 170 f .005 .005 .005 .012 .015 .030 .047 .095 .162 .284 L 10 11 12 13 14 15 16 17 18 19 N 62 54 77 53 46 48 30 21 16 11 SN 232 286 363 416 462 510 540 561 577 588 f .388 .478 .607 .696 .773 .853 .903 .938 .965 .983 L 20 21 22 23 24 25 26 27 28 29 N 4 1 2 1 0 0 0 0 1 0 SN 592 593 595 596 596 596 596 596 597 597 f .990 .992 .995 .997 .997 .997 .997 .997 .998 .998 L 30 31 32 33 34 35 36 37 38 39 N 0 0 0 0 0 0 0 0 0 0 SN 597 597 597 597 597 597 597 597 597 597 f .998 .998 .998 .998 .998 .998 .998 .998 .998 .998 L 40 41 42 N 0 0 1 SN 597 597 598 f .998 .998 1.0 Median = 11.17 L is the length N is the number of examples Sum(N) = SN is the sum of the Ns f is the cumulative fraction seen

TABLE 9P Tally of left-aligned CDR3 sequences A C D E F G H I K L M # 1 74 6 278 109 11 319 50 18 11 60 8 1383 GDERVASLHTNQPIWYFKMCX 2 50 9 64 32 29 249 43 42 41 109 22 1377 GRPSLDVYTANHIQKEFMWCX 3 81 18 74 39 25 214 29 42 16 83 19 1377 GSYRTVLADPIWEQHNFMCK| 4 70 23 92 49 50 228 23 58 21 70 16 1373 GSYDRVALTIPFEWNCHQKMX 5 86 28 106 32 59 217 21 41 16 72 19 1371 GYSDAVTLRFIPWNECHMQK|X 6 88 17 104 28 94 171 17 48 12 50 17 1362 GYSDFATVRWPLINEQCHMK| 7 69 15 110 21 89 176 22 50 15 81 12 1349 GSYDFVLTAPRWINHEQCKM|X 8 53 19 141 17 90 150 18 47 17 68 11 1311 YSGDFLTVWAPIRNCHEKQM| 9 44 21 120 24 102 174 24 36 20 71 11 1250 YGSDFLNVRTAWPIEHCKQM| 10 39 31 129 23 124 116 23 42 9 58 32 1162 YDFGSLIARPTVWNMCEHQK 11 36 12 158 17 137 83 13 18 10 40 21 1061 YDFGSPLVANWMTRIEHCKQX 12 34 11 164 10 82 74 34 30 1 31 20 943 YDFGPSVAHLINMRTWCEQKX 13 32 2 121 6 84 56 10 26 7 43 32 789 YDFGLSPVAMIWRTHNKQEC 14 23 131 5 59 65 10 16 4 25 34 639 YDGFMVLAPISWNRHTQEKX 15 15 4 107 5 43 42 1 23 20 34 521 YDFGVMILWAPRSENCQTH| 16 4 2 80 3 33 26 4 5 1 10 29 396 YDVFMGPSLNTRIWAHECQ|K 17 3 1 63 19 19 9 13 12 21 291 DYVMFGILHPSTWAQRCNX 18 3 47 16 13 1 4 7 23 207 DYVMFGPSLTIAHN 19 5 1 39 1 4 13 3 3 1 14 146 DYVMGAFHINRSCELPQW 20 2 17 4 5 3 4 12 100 VYDMGFLIPSARWQ 21 17 3 8 1 1 4 58 DVGYMFHINTW 22 1 7 6 1 1 5 42 VDFMYSAGITW 23 9 1 1 1 1 25 DVYGILMPS 24 1 2 1 1 1 18 VYDAHLMPT 25 1 3 9 GVDPSY 26 2 2 7 GMSTV 27 2 1 1 6 DKMST 28 1 1 1 6 VADGS 29 1 4 DPSV 30 1 3 FST 31 1 1 3 KLV 32 1 1 3 FGP 33 1 3 PG 34 1 1 3 HLS 35 1 3 AVW 36 1 1 3 DFP 37 3 PSY 38 1 2 LS 39 1 1 2 AK 40 2 PS 41 2 ST 42 2 S 43 1 1 K 44 1 S 45 1 T 46 1 S 816 220 2186 421 1166 2428 358 568 205 920 421 N P Q R S T V W Y | X # 1 35 23 31 108 63 50 94 16 13 6 1383 GDERVASLHTNQPIWYFKMCX 2 44 114 42 169 114 59 62 21 60 2 1377 GRPSLDVYTANHIQKEFMWCX 3 26 73 37 110 140 97 89 42 122 1 1377 GSYRTVLADPIWEQHNFMCK| 4 48 51 22 79 141 65 77 49 139 2 1373 GSYDRVALTIPFEWNCHQKMX 5 37 41 18 61 157 75 85 38 158 2 2 1371 GYSDAVTLRFIPWNECHMQK|X 6 32 54 23 67 152 80 78 64 165 1 1362 GYSDFATVRWPLINEQCHMK| 7 44 59 18 58 157 73 85 54 139 1 1 1349 GSYDFVLTAPRWINHEQCKM|X 8 38 48 14 41 167 68 59 59 185 1 1311 YSGDFLTVWAPIRNCHEKQM| 9 52 40 14 47 123 45 48 41 192 1 1250 YGSDFLNVRTAWPIEHCKQM| 10 33 37 12 39 73 36 36 35 235 1162 YDFGSLIARPTVWNMCEHQK 11 33 49 7 20 68 21 37 29 251 1 1061 YDFGSPLVANWMTRIEHCKQX 12 30 53 10 19 45 19 42 18 215 1 943 YDFGPSVAHLINMRTWCEQKX 13 10 34 7 22 40 15 33 25 184 789 YDFGLSPVAMIWRTHNKQEC 14 13 22 6 12 15 10 26 14 148 1 639 YDGFMVLAPISWNRHTQEKX 15 5 12 3 12 12 3 40 20 119 1 521 YDFGVMILWAPRSENCQTH| 16 10 24 2 6 12 7 49 5 82 2 396 YDVFMGPSLNTRIWAHECQ|K 17 1 8 2 2 8 5 42 4 58 1 291 DYVMFGILHPSTWAQRCNX 18 1 13 8 5 31 35 207 DYVMFGPSLTIAHN 19 2 1 1 2 2 24 1 29 146 DYVMGAFHINRSCELPQW 20 3 1 2 3 23 2 19 100 VYDMGFLIPSARWQ 21 1 1 14 1 7 58 DVGYMFHINTW 22 2 1 12 1 5 42 VDFMYSAGITW 23 1 1 5 5 25 DVYGILMPS 24 1 1 5 5 18 VYDAHLMPT 25 1 1 2 1 9 GVDPSY 26 1 1 1 7 GMSTV 27 1 1 6 DKMST 28 1 2 6 VADGS 29 1 1 1 4 DPSV 30 1 1 3 FST 31 1 3 KLV 32 1 3 FGP 33 2 3 PG 34 1 3 HLS 35 1 1 3 AVW 36 1 3 DFP 37 1 1 1 3 PSY 38 1 2 LS 39 2 AK 40 1 1 2 PS 41 1 1 2 ST 42 2 2 S 43 1 K 44 1 1 S 45 1 1 T 46 1 1 S 495 769 270 876 1518 741 1104 540 2572 10 17 18621

TABLE 10P Tally of right-aligned sequences A C D E F G H I K L M # 5 1 1 G 6 1 S 7 1 1 G 8 1 1 G 9 2 RV 10 2 RV 11 1 1 2 GI 12 2 V 13 2 TY 14 1 1 3 DGN 15 1 3 ISY 16 1 3 DSY 17 1 3 APY 18 1 1 1 3 DFM 19 2 1 3 DG 20 1 1 3 ILV 21 3 WP 22 3 4 GS 23 2 1 6 GHQSV 24 1 3 1 6 GALR 25 1 2 1 7 DTAIS 26 1 1 1 1 1 1 1 9 ACDGKLMST 27 2 5 1 2 1 1 18 DAGVEILNQRS 28 2 2 3 1 2 25 TGQSDELPRIV 29 3 5 6 7 1 1 1 42 GEDVAPQRSKLMTY| 30 2 9 1 9 1 4 5 2 58 DGRLSIVPAMQTFHNY 31 4 2 19 9 2 18 1 2 1 3 100 DGSERVYALPTCFINHKW 32 10 5 18 5 3 16 3 3 2 14 1 146 DGLRVAPYSTCEQFHINWKM 33 20 18 10 7 34 7 8 2 6 1 207 GARDPSYTEVIFHLQWKM 34 13 4 31 18 9 37 8 16 4 14 4 291 GDRYPVEILASTFHQWCKMNX| 35 17 5 32 23 10 70 12 10 6 25 1 396 GRSDYLEVTPAHNFIWKCQM| 36 23 6 51 21 9 79 19 15 14 36 9 521 GDSYRLTVPAEHIKNFMWCQ| 37 35 12 56 23 15 110 14 17 5 24 4 639 GYDVRSTAPLEIFHNCWQKMX 38 28 19 68 27 29 133 26 31 12 43 7 789 GSYDVRLPTIFAEHCNWKQM 39 51 25 80 27 33 162 16 30 18 55 15 943 GSDRYVLATPFWIECKHMQNX 40 44 14 73 36 46 161 27 32 17 59 8 1061 GSRDYVTLPFAEIWHQNKCM 41 54 21 74 25 23 178 23 52 15 57 11 1162 GSYTDRVLPAIWNQEFHCKMX| 42 57 13 82 40 42 190 14 39 15 82 15 1250 GSYDLVRTANPFEIWQKMHC| 43 75 18 54 25 35 242 13 29 18 49 12 1311 GYSTARVPDLWNFIQECKHM| 44 63 17 79 15 43 197 20 38 14 76 8 1349 YGSTDLRAPVWNFIQHCEKM 45 59 16 69 35 55 165 26 23 23 75 9 1362 YGSLRTDNAFPVWEHIKCQM 46 41 19 125 26 27 208 31 14 16 38 8 1371 YGDSNRWATLPHFEVQCKIM 47 160 10 24 13 53 332 36 16 11 40 10 1373 GYAWPSFRLHTVNDIEKCMQX 48 21 4 8 5 680 27 4 44 5 145 288 1377 FMLISGVYPAWTDNQREKCHX 49 23 2 1181 29 1 30 15 4 2 8 1 1377 DGEAHNQSYVLPTIRCKW|FMX 50 7 7 15 42 3 41 135 3 59 4 1383 YVIPSLFHNDTACXMGKQRW| 816 220 2186 421 1166 2428 358 568 205 920 421 N P Q R S T V W Y | X # 5 1 G 6 1 1 S 7 1 G 8 1 G 9 1 1 2 RV 10 1 1 2 RV 11 2 GI 12 2 2 V 13 1 1 2 TY 14 1 3 DGN 15 1 1 3 ISY 16 1 1 3 DSY 17 1 1 3 APY 18 3 DFM 19 3 DG 20 1 3 ILV 21 1 2 3 WP 22 1 4 GS 23 1 1 1 6 GHQSV 24 1 6 GALR 25 1 2 7 DTAIS 26 1 1 9 ACDGKLMST 27 1 1 1 1 2 18 DAGVEILNQRS 28 2 3 2 3 4 1 25 TGQSDELPRIV 29 3 3 2 2 1 5 1 1 42 GEDVAPQRSKLMTY| 30 1 3 2 7 5 2 4 1 58 DGRLSIVPAMQTFHNY 31 2 3 7 10 3 7 1 6 100 DGSERVYALPTCFINHKW 32 3 9 4 12 8 6 12 3 9 146 DGLRVAPYSTCEQFHINWKM 33 16 6 19 15 12 10 3 13 207 GARDPSYTEVIFHLQWKM 34 2 20 5 31 12 12 20 5 23 1 2 291 GDRYPVEILASTFHQWCKMNX| 35 12 18 5 39 35 19 23 7 26 1 396 GRSDYLEVTPAHNFIWKCQM| 36 11 24 6 42 47 29 28 7 44 1 521 GDSYRLTVPAEHIKNFMWCQ| 37 14 33 9 54 52 37 55 11 58 1 639 GYDVRSTAPLEIFHNCWQKMX 38 18 33 12 46 77 32 58 17 73 789 GSYDVRLPTIFAEHCNWKQM 39 11 38 12 70 94 42 61 33 68 2 943 GSDRYVLATPFWIECKHMQNX 40 24 52 27 74 140 61 66 29 71 1061 GSRDYVTLPFAEIWHQNKCM 41 31 55 29 70 156 76 61 51 97 1 2 1162 GSYTDRVLPAIWNQEFHCKMX| 42 48 47 24 68 171 68 70 39 125 1 1250 GSYDLVRTANPFEIWQKMHC| 43 38 58 28 73 164 76 66 43 194 1 1311 GYSTARVPDLWNFIQECKHM| 44 48 60 24 69 131 86 57 52 252 1349 YGSTDLRAPVWNFIQHCEKM 45 62 51 16 75 116 74 50 39 324 1362 YGSLRTDNAFPVWEHIKCQM 46 97 38 21 55 110 39 26 55 377 1371 YGDSNRWATLPHFEVQCKIM 47 25 54 9 44 54 34 32 122 292 2 1373 GYAWPSFRLHTVNDIEKCMQX 48 8 22 7 6 28 10 25 16 23 1 1377 FMLISGVYPAWTDNQREKCHX 49 15 6 13 4 13 5 9 2 11 2 1 1377 DGEAHNQSYVLPTIRCKW|FMX 50 23 122 3 3 67 9 350 3 480 1 6 1383 YVIPSLFHNDTACXMGKQRW| 50 495 769 270 876 1518 741 1104 540 2572 10 17 18621

TABLE 11P Tallies of AA frequencies in all CDR3 by length Tally of sequences of length 7 # = 38 A C D E F G H I K L M # 1 1 8 1 1 14 1 1 5 38 GDLRWAEFHKS 2 1 1 2 6 3 2 1 1 38 RGNHVFKTYADLMW 3 1 4 1 5 1 2 2 38 GSDWYPVILTAFHN 4 3 1 1 12 1 1 1 38 GYSANRVDFHILPT 5 2 1 14 3 4 1 3 3 38 FIGLMARVYEKP 6 26 1 1 38 DVPTHISWY 7 1 2 2 3 1 38 YVINDHSALR 9 42 2 19 40 9 11 4 13 4 N P Q R S T V W Y | X # 1 3 1 2 38 GDLRWAEFHKS 2 6 7 2 3 1 2 38 RGNHVFKTYADLMW 3 1 3 5 2 3 4 4 38 GSDWYPVILTAFHN 4 2 1 2 4 1 2 6 38 GYSANRVDFHILPT 5 1 2 2 2 38 FIGLMARVYEKP 6 2 1 2 3 1 1 38 DVPTHISWY 7 3 1 2 7 16 38 YVINDHSALR 12 7 15 13 7 20 8 31 266 Tally of sequences of length 8 # = 61 A C D E F G H I K L M # 1 3 7 3 14 2 2 5 61 GDLTVRSAEHINWPQY 2 1 9 1 1 15 1 2 1 61 GDTNRSVKWYAEFILPQ 3 2 3 1 10 1 1 7 1 61 GLSTYVDPRAFHIMNQW 4 4 1 3 1 1 15 1 4 61 GYRALQDSWVCEFHNPT 5 10 2 1 9 5 1 5 1 61 AGYHLTPRVDSEKMW 6 5 1 24 2 7 5 2 61 FIALPSVYGMCQRW 7 5 37 2 4 1 2 61 DAHSELNVIP| 8 1 2 3 1 12 3 61 YISFLVDNAHPRT 31 2 63 8 30 65 14 24 3 32 4 N P Q R S T V W Y | X # 1 2 1 1 4 4 5 5 2 1 61 GDLTVRSAEHINWPQY 2 6 1 1 4 3 8 3 2 2 61 GDTNRSVKWYAEFILPQ 3 1 3 1 3 7 7 5 1 7 61 GLSTYVDPRAFHIMNQW 4 1 1 4 5 3 1 2 3 11 61 GYRALQDSWVCEFHNPT 5 4 4 2 5 4 1 7 61 AGYHLTPRVDSEKMW 6 3 1 1 3 3 1 3 61 FIALPSVYGMCQRW 7 2 1 4 2 1 61 DAHSELNVIP| 8 2 1 1 7 1 3 24 61 YISFLVDNAHPRT 14 15 8 22 33 27 27 10 55 1 488 Tally of sequences of length 9 # = 88 A C D E F G H I K L M # 1 9 12 4 21 1 1 2 5 88 GDARNVLEQTKWHIPSY 2 2 2 3 3 13 4 3 7 2 88 GPSRLNTHEFKYADMQW 3 4 2 3 3 3 15 1 1 88 GTPSQNRVWYADEFCLM 4 5 1 6 3 6 22 2 4 1 6 1 88 GSDFLARITYENPWHVCKM 5 7 1 4 3 4 14 2 7 2 88 GSYALNDFVERWHMQTCP 6 13 2 1 3 13 6 2 1 4 1 88 YAGHNLPSVFTWDIEKMQR 7 4 2 41 2 3 1 14 5 88 FLMAPWIDGSVKNQTY 8 1 1 73 2 2 1 2 88 DEGLSACHNQRV 9 1 1 4 1 3 8 2 88 YVISFHPLNTCDGR 45 6 105 19 64 103 19 18 8 48 12 N P Q R S T V W Y | X # 1 7 1 3 8 1 3 7 2 1 88 GDARNVLEQTKWHIPSY 2 5 11 2 10 11 5 2 3 88 GPSRLNTHEFKYADMQW 3 5 7 6 5 7 11 5 5 5 88 GTPSQNRVWYADEFCLM 4 3 3 5 7 4 2 3 4 88 GSDFLARITYENPWHVCKM 5 6 1 2 3 12 2 4 3 11 88 GSYALNDFVERWHMQTCP 6 5 4 1 1 4 3 4 3 17 88 YAGHNLPSVFTWDIEKMQR 7 1 4 1 2 1 2 4 1 88 FLMAPWIDGSVKNQTY 8 1 1 1 2 1 88 DEGLSACHNQRV 9 2 3 1 8 2 9 43 88 YVISFHPLNTCDGR 35 34 16 34 54 31 34 22 85 792 Tally of sequences of length 10 # = 101 A C D E F G H I K L M # 1 8 1 19 7 1 16 3 2 3 2 101 DGNAERTSQVHLWKMYCF 2 3 8 3 5 13 5 15 2 101 LGRDSPVFINTAEQYMW 3 6 9 1 26 1 3 1 4 1 101 GSYDAVTLNRIPWFHKMQ 4 7 6 1 25 1 5 4 1 101 GSYARDINPLTVWQFHM 5 6 5 9 4 16 1 3 4 101 GYTESANDPRFLVKQWH 6 6 1 6 5 4 23 2 4 3 3 1 101 GYRSWADEFINKLTHCMQV 7 13 3 1 5 9 3 1 4 1 101 YASGPRWFTVLDHNEIMQ 8 2 1 1 57 3 4 15 4 101 FLIMSGWANPVCEY 9 3 78 2 6 1 1 1 101 DGAQENIKLPRSW 10 3 4 4 13 1 101 YIPSVFHNDL 54 3 137 28 82 137 15 36 10 54 12 N P Q R S T V W Y | X # 1 9 4 6 5 6 4 3 2 101 DGNAERTSQVHLWKMYCF 2 5 6 3 11 8 4 6 1 3 101 LGRDSPVFINTAEQYMW 3 4 3 1 4 14 5 6 2 10 101 GSYDAVTLNRIPWFHKMQ 4 5 5 3 7 11 4 4 4 8 101 GSYARDINPLTVWQFHM 5 6 5 2 5 8 10 4 2 11 101 GYTESANDPRFLVKQWH 6 4 1 8 7 3 1 7 12 101 GYRSWADEFINKLTHCMQV 7 2 7 1 7 11 5 5 6 17 101 YASGPRWFTVLDHNEIMQ 8 2 2 4 2 3 1 101 FLIMSGWANPVCEY 9 2 1 3 1 1 1 101 DGAQENIKLPRSW 10 4 8 7 5 52 101 YIPSVFHNDL 43 37 18 49 76 37 37 29 116 1010 Tally of sequences of length 11 # = 118 A C D E F G H I K L M # 1 7 1 21 11 23 5 2 7 118 GDEVRALQHSPTINCWY 2 1 2 9 1 1 24 5 6 2 7 3 118 GSRDYLPIVHQTMNCKWAEFX 3 4 4 2 4 13 2 3 1 7 2 118 SGTVRLYWADFNQIEHMKP 4 10 3 3 2 25 1 2 4 3 118 SGARTWYLVDEMQFINPH 5 5 2 10 1 4 24 2 1 5 1 118 GSVYDTNALRFWCHQEKM 6 6 4 2 7 19 2 3 1 5 1 118 GSYWTFAVLRDINEHQKMP 7 4 1 8 5 2 20 4 1 2 1 118 GYSNRDWTEPAHFLQVCIM 8 13 2 6 1 8 12 4 2 7 118 YAGWFLDPRSTHCKVE 9 2 2 68 2 5 14 7 118 FLMYVITADGP 10 2 1 100 5 3 2 1 1 118 DEGAHCLMNPQ 11 2 6 1 7 1 6 1 118 YPVISFLNDHKM 54 9 169 31 102 165 28 29 8 65 20 N P Q R S T V W Y | X # 1 2 4 7 8 5 3 10 1 1 118 GDEVRALQHSPTINCWY 2 3 7 4 10 11 4 6 2 9 1 118 GSRDYLPIVHQTMNCKWAEFX 3 4 1 4 8 25 12 9 6 7 118 SGTVRLYWADFNQIEHMKP 4 2 2 3 9 26 8 4 6 5 118 SGARTWYLVDEMQFINPH 5 6 2 5 15 9 11 4 11 118 GSVYDTNALRFWCHQEKM 6 3 1 2 5 16 9 6 11 15 118 GSYWTFAVLRDINEHQKMP 7 9 5 2 9 11 6 2 7 19 118 GYSNRDWTEPAHFLQVCIM 8 6 5 5 5 2 11 29 118 YAGWFLDPRSTHCKVE 9 1 4 6 7 118 FLMYVITADGP 10 1 1 1 118 DEGAHCLMNPQ 11 3 13 7 11 60 118 YPVISFLNDHKM 33 41 25 59 121 60 67 48 163 1 1298 Tally of sequences of length 12 # = 154 A C D E F G H I K L M # 1 5 31 12 37 6 1 1 7 3 154 GDRESVLHAPMNQTWYIK 2 5 1 7 6 1 25 3 7 3 13 2 154 GSRLPDIQEAVYHKNTMWCF 3 10 2 7 5 1 19 5 4 12 2 154 GRSYLATVPDQEIKWCMNF 4 8 9 6 8 27 6 5 6 1 154 GVSDNAFRTYEILKWPQM 5 18 1 8 5 6 42 1 9 1 7 3 154 GSAIDYLFPTEQVMNWCHK 6 13 12 4 10 23 1 7 8 1 154 GAVDSFYTLPRWINEQHM 7 11 2 4 3 10 15 1 4 12 154 YGSPLRAFWTNVDIECQH 8 3 2 18 3 3 25 4 2 5 6 154 YGDSNLTKRWHPAEFCIQV 9 15 1 2 8 33 4 7 1 5 1 154 GYWARFISPLHTDQCKMN 10 1 1 2 1 79 1 2 5 1 19 26 154 FMLIPYDHVWACEGKNQRST 11 2 135 2 4 2 154 DGYAEHSVNR 12 1 1 6 1 9 16 4 154 YVPIHFSLNCDGW 91 11 236 47 132 252 33 69 21 99 39 N P Q R S T V W Y | X # 1 3 4 3 14 10 3 10 2 2 154 GDRESVLHAPMNQTWYIK 2 3 11 7 22 24 3 5 2 4 154 GSRLPDIQEAVYHKNTMWCF 3 2 8 6 17 17 9 9 4 15 154 GRSYLATVPDQEIKWCMNF 4 9 4 4 7 17 7 18 5 7 154 GVSDNAFRTYEILKWPQM 5 3 6 4 20 6 4 2 8 154 GSAIDYLFPTEQVMNWCHK 6 5 8 3 8 11 9 13 8 10 154 GAVDSFYTLPRWINEQHM 7 5 14 2 12 15 6 5 9 24 154 YGSPLRAFWTNVDIECQH 8 10 4 2 5 15 6 2 5 34 154 YGDSNLTKRWHPAEFCIQV 9 1 6 2 10 7 3 18 30 154 GYWARFISPLHTDQCKMN 10 1 4 1 1 1 1 2 2 3 154 FMLIPYDHVWACEGKNQRST 11 1 1 2 2 3 154 DGYAEHSVNR 12 2 18 5 32 1 58 154 YVPIHFSLNCDGW 45 87 34 97 144 53 102 58 198 1848 Tally of sequences of length 13 # = 150 A C D E F G H I K L M # 1 4 2 28 9 3 37 8 3 3 5 150 GDTESHRVLPAQFIKCNW 2 11 4 4 1 2 32 3 1 5 11 3 150 GRSPALTKVCDYHMQWFEIN 3 7 2 8 4 4 23 11 1 4 6 2 150 GSYHQTDPRAVLEFKNCMWI 4 6 2 6 4 6 30 1 8 6 1 150 GSWYTIADFLPVEQRCHMNX 5 8 10 4 2 28 1 2 22 3 150 GLSYDATWPREQMNVFIH 6 10 2 11 1 6 21 2 2 5 1 150 GYSPTDAQVFRLNWCIKEM 7 5 1 8 1 4 19 1 6 5 21 2 150 LGYSTDPIRVAKFNWMQCEH 8 7 5 22 5 3 12 3 3 3 8 1 150 YDSGLARTCEQVNPFHIKWM 9 1 2 12 3 1 26 7 2 4 7 2 150 NGYDSWHLPRKETVCIMAFQ 10 19 1 2 2 17 24 5 2 5 1 150 YGAFWHLPTNSVDEIQRCM 11 1 1 105 2 2 1 13 14 150 FMLYGIVAEKPQRSWX 12 130 3 5 1 150 DGYEQNHT 13 1 2 5 5 14 18 1 150 YVLIPSFHTDAMN 80 21 243 38 158 259 46 46 27 127 31 N P Q R S T V W Y | X # 1 2 5 4 8 9 11 8 1 150 GDTESHRVLPAQFIKCNW 2 1 13 3 20 17 7 5 3 4 150 GRSPALTKVCDYHMQWFEIN 3 3 8 11 8 16 11 7 2 12 150 GSYHQTDPRAVLEFKNCMWI 4 1 6 4 4 18 10 6 16 14 1 150 GSWYTIADFLPVEQRCHMNX 5 3 6 4 5 19 8 3 7 15 150 GLSYDATWPREQMNVFIH 6 3 15 8 6 16 13 8 3 17 150 GYSPTDAQVFRLNWCIKEM 7 4 7 2 6 15 14 6 4 19 150 LGYSTDPIRVAKFNWMQCEH 8 4 4 5 7 15 7 5 2 29 150 YDSGLARTCEQVNPFHIKWM 9 31 5 1 5 10 3 3 9 16 150 NGYDSWHLPRKETVCIMAFQ 10 3 5 2 2 3 4 3 15 35 150 YGAFWHLPTNSVDEIQRCM 11 1 1 1 1 2 1 3 1 150 FMLYGIVAEKPQRSWX 12 2 3 1 5 150 DGYEQNHT 13 1 14 13 4 21 51 150 YVLIPSFHTDAMN 58 89 48 72 152 93 77 63 220 2 1950 Tally of sequences of length 14 # = 118 A C D E F G H I K L M # 1 6 29 7 2 32 8 1 1 2 118 GDVHERTAFLPSIKNQ 2 4 10 1 5 22 7 3 4 7 118 GPDRYSVHLFAKIQTENW 3 11 2 7 2 3 25 5 1 9 2 118 GVARYLSDITFWCEMPK 4 5 2 7 7 3 12 4 4 3 6 118 SGVYPDELRTANHIFKWC 5 6 5 12 2 18 2 2 2 4 1 118 GYSDTVARCLPFHIKNWMQ 6 6 10 5 4 16 5 3 2 1 118 YGSTDRAEIFVKWLPQMN 7 4 4 1 4 32 2 2 2 1 118 GSVTYNADFHIKPQRWEM 8 6 1 5 1 4 18 2 5 3 2 118 GSYTWAPRDIFNVLHMCE 9 5 2 4 1 2 11 2 1 5 9 1 118 YSGTLVAKNRDWCFHPEIM 10 2 5 9 2 3 21 2 2 4 118 YGSDNTCQLRFWAEIKPV 11 12 1 3 5 25 2 2 1 118 YGWAPVFNEHLTDMQR 12 1 64 5 1 5 12 16 118 FMLGIPSVAHQTY 13 3 97 4 5 1 1 1 1 118 DGEANQHIKLV 14 2 3 4 12 6 118 YVPILHFANS 73 17 195 34 104 242 35 48 24 67 25 N P Q R S T V W Y | X # 1 1 2 1 7 2 7 10 118 GDVHERTAFLPSIKNQ 2 1 13 2 10 8 2 8 1 10 118 GPDRYSVHLFAKIQTENW 3 2 11 8 4 13 3 10 118 GVARYLSDITFWCEMPK 4 5 8 6 13 6 12 3 12 118 SGVYPDELRTANHIFKWC 5 2 3 1 6 15 10 7 2 18 118 GYSDTVARCLPFHIKNWMQ 6 1 2 2 7 16 12 4 3 19 118 YGSTDRAEIFVKWLPQMN 7 5 2 2 2 18 12 13 2 10 118 GSVTYNADFHIKPQRWEM 8 4 6 6 16 12 4 9 14 118 GSYTWAPRDIFNVLHMCE 9 5 2 5 14 10 8 4 27 118 YSGTLVAKNRDWCFHPEIM 10 6 2 5 4 13 6 2 3 27 118 YGSDNTCQLRFWAEIKPV 11 4 7 1 1 2 6 14 32 118 YGWAPVFNEHLTDMQR 12 4 1 4 1 3 1 118 FMLGIPSVAHQTY 13 2 2 1 118 DGEANQHIKLV 14 2 14 2 20 53 118 YVPILHFANS 38 67 17 65 129 84 111 44 233 1652 Tally of sequences of length 15 # = 125 A C D E F G H I K L M # 1 7 26 8 3 29 1 3 10 125 GDLREASTVNFIPYH 2 6 2 3 22 3 4 1 9 125 RGPLNSTYAVIQEHWDK 3 4 4 5 7 2 19 2 6 2 9 2 125 GRYLSVEPIDTACQWFHKMN 4 7 4 14 6 6 15 2 7 5 7 4 125 GDYAILVEFRKSTCMNPWHQ 5 6 3 10 2 5 18 4 2 3 2 125 GSYVDRWAFTICLNEKMP 6 6 2 7 2 5 10 1 5 7 1 125 SRYGTDLWAPFIVNCEQHM 7 8 4 14 2 2 22 3 3 1 9 1 125 GSDLAVRPYCTHIWEFNKM 8 6 2 4 22 2 2 3 125 GYSVWRATDNPLCIKQ 9 4 3 8 4 20 4 3 1 6 125 YGSDLPTRVAFHQCINKW 10 3 4 5 8 8 17 1 3 7 125 YGEFNTLSRDVCPAIWH 11 4 2 15 3 3 17 1 1 1 125 YGDSNPAWEFRTCQHIKV 12 22 3 2 31 3 1 3 3 125 GYAWPSNCHLMFQRVITX 13 71 1 4 6 30 125 FMLISQTVGPRY 14 115 2 1 1 1 125 DNEFGHPQ 15 3 5 1 1 20 7 1 125 YVILPFSCNGHMQ 83 34 225 43 117 245 23 66 15 86 44 N P Q R S T V W Y | X # 1 4 3 10 7 6 6 2 125 GDLREASTVNFIPYH 2 8 11 4 23 7 7 5 3 7 125 RGPLNSTYAVIQEHWDK 3 2 7 3 13 9 5 8 3 13 125 GRYLSVEPIDTACQWFHKMN 4 4 4 1 6 5 5 7 3 13 125 GDYAILVEFRKSTCMNPWHQ 5 3 2 8 18 5 11 8 15 125 GSYVDRWAFTICLNEKMP 6 3 6 2 12 24 9 4 7 12 125 SRYGTDLWAPFIVNCEQHM 7 2 6 7 21 4 8 3 5 125 GSDLAVRPYCTHIWEFNKM 8 4 4 2 7 19 5 12 10 21 125 GYSVWRATDNPLCIKQ 9 3 6 4 5 19 6 5 1 23 125 YGSDLPTRVAFHQCINKW 10 8 4 6 7 8 5 2 29 125 YGEFNTLSRDVCPAIWH 11 7 5 2 3 14 3 1 4 39 125 YGDSNPAWEFRTCQHIKV 12 4 7 2 2 6 1 2 8 24 1 125 GYAWPSNCHLMFQRVITX 13 1 2 1 4 2 2 1 125 FMLISQTVGPRY 14 3 1 1 125 DNEFGHPQ 15 2 7 1 5 33 39 125 YVILPFSCNGHMQ 57 74 24 103 165 66 109 52 243 1 1875 Distribution of D-JH with number of cys's 0 1 2 3 4 1248 53 80 1 1 Tally of AAs in the YYCar motif A C D E F G H I K L M # 1 1 1 14 1 1383 YFDEH 2 4 1 92 11 4 1383 YFHCLSWDR 3 1379 1383 CRS 4 1207 3 2 12 2 2 1383 AVTSGNDFILRQX 5 14 1 4 18 17 9 187 4 1 1383 RKTSGHAIVNFLQYPEM| 1221 1383 5 2 112 30 29 11 187 10 1 N P Q R S T V W Y | X # 1 1366 1383 YFDEH 2 1 3 2 1265 1383 YFHCLSWDR 3 2 2 1383 CRS 4 4 1 2 17 51 79 1 1383 AVTSGNDFILRQX 5 7 2 3 992 55 56 9 3 1 1383 RKTSGHAIVNFLQYPEM| 11 2 4 997 77 107 88 2 2634 1 1 6915

TABLE 12P Alignment and tabulation of sequences having 3-22 D segments D3:3-22_Phz0 YYYDSSGYYY = GLG Entry Seq1 L1 Seq2 L2 JH P Score 1 hs3d6hcv GRDYYDSGGYFT 12 GRDYYDSGGYFTVAFDI 17 3 6 1.76D+13 2 hs6d4xb7 DRHNYYDSSGSYS 13 DRHNYYDSSGSYSDY 15 4 9 4.40D+12 3 hs6d4xg3 DCPAPAKMYYYGSGICT 17 DCPAPAKMYYYGSGICTFDY 20 4 3 6.55D+04 4 hs83x6f2 AFYDSAD 7 AFYDSADDY 9 4 −4 2.62D+05 5 hsa230644 RDYYDSSGPEAG 12 RDYYDSSGPEAGFDI 15 3 3 6.87D+10 6 hsa239386 DGTLIDTSAYYYL 13 DGTLIDTSAYYYLY 14 4 6 6.87D+10 7 hsa234232 NSSDSS 6 NSSDSSVLDV 10 6 −4 6.55D+04 8 hsa239378 DQVFDSGGYNHR 12 DQVFDSGGYNHRFDS 15 4 3 1.07D+09 9 hsa239367 DLEYYYDSGGHYSP 14 DLEYYYDSGGHYSPFHY 17 4 9 1.10D+12 10 hsa239339 DDSSGY 6 DDSSGYYYIDY 11 4 −10 1.72D+10 11 hsa245311 GHYYDSPGQYSYS 13 GHYYDSPGQYSYSEY 15 4 3 1.07D+09 12 hsa240578 GGFRPPPYDYESSAYRTYR 19 GGFRPPPYDYESSAYRTYRLDF 22 4 21 2.75D+11 13 hsa245359 DSDTRAY 7 DSDTRAYYWYFDL 13 2 −7 1.68D+07 14 hsa245028 GRHYYDSSGYYSTPE 15 GRHYYDSSGYYSTPENYFDY 20 4 6 1.80D+16 15 hsa245019 DPSYYYDSSGLPL 13 DPSYYYDSSGLPLHGMDV 18 6 9 4.40D+12 16 hsa244991 TYYYDSSGYLLTR 13 TYYYDSSGYLLTRYFQH 17 1 3 4.50D+15 17 hsa244945 NAPHYDSSGYYQT 13 NAPHYDSSGYYQTFDY 16 4 6 7.04D+13 18 hsa244943 GYHSSSYA 8 GYHSSSYADAFDI 13 3 −7 6.71D+07 19 hsa245289 PIGYCSGGSC 10 PIGYCSGGSCYSFDY 15 4 −4 2.62D+05 20 hsa240554 THGTYVTSGYYPKI 14 THGTYVTSGYYPKI 14 4 6 2.68D+08 21 hsa279533 GATYYYESSGNYP 13 GATYYYESSGNYPDY 15 4 9 7.04D+13 22 hsa389177 AFYHYDSTGYPNRRY 15 AFYHYDSTGYPNRRYYFDY 19 4 6 4.29D+09 23 hsa7321 SYSYYYDSSGYWGG 14 SYSYYYDSSGYWGGYFDY 18 4 9 4.50D+15 24 hsaj2772 LSPYYYDSSSYH 12 LSPYYYDSSSYHDAFDI 17 3 6 2.62D+05 25 hsb7g4f08 EEDYYDSSGQAS 12 EEDYYDSSGQASYNWFXP 18 5 6 2.75D+11 26 hsb7g3b02 ETNYYDSGGYPG 12 ETNYYDSGGYPGFDF 15 4 6 4.40D+12 27 hsb7g3c12 GDHYYDRSGYRH 12 GDHYYDRSGYRHSYYYYAMDV 21 6 6 2.75D+11 28 hsb8g3b07 DRSSGN 6 DRSSGNYFDGMDV 13 6 −10 6.55D+04 29 hsfog1h GRSRYSGYG 9 GRSRYSGYGFYSGMDV 16 6 −4 2.62D+05 30 hsgvh0209 DDTSGYGP 8 DDTSGYGPYYFYYGMDV 17 6 −10 2.68D+08 31 hsgvh55 RAYYDTSFYFEY 12 RAYYDTSFYFEYY 13 4 3 1.72D+10 32 hsgvh0304 DRIDYYKSGYYLGSA 15 DRIDYYKSGYYLGSADS 17 4 6 1.68D+07 33 hsgvh0332 DTDSSSHYG 9 DTDSSSHYGRFDP 13 5 −7 1.68D+07 34 hsgvh0328 VSISHYDSSGRPQRVF 16 VSISHYDSSGRPQRVFYGMDV 21 6 9 1.07D+09 35 hsgvh536 QARENVFYDSSGPTAP 16 QARENVFYDSSGPTAPFDH 19 4 15 1.72D+10 36 hshcmg42 VPAGNYYDTSGPDN 14 VPAGNYYDTSGPDNAD 16 4 12 1.72D+10 37 hsig001vh WYYFDTSGYYPRNFYYMDV 19 WYYFDTSGYYPRNFYYMDV 19 4 3 2.81D+14 38 hsig13g10 GYYYDSGGNYNG 12 GYYYDSGGNYNGDY 14 4 3 1.10D+12 39 hsighpat3 DLRSYDPSGYYN 12 DLRSYDPSGYYNDGFDI 17 3 6 2.75D+11 40 hsigh13g7 GYYYDRGGNCNG 12 GYYYDRGGNCNGDY 14 4 3 6.87D+10 41 hsigh13g1 GYYYDRGGNYNG 12 GYYYDRGGNYNGDY 14 4 3 1.10D+12 42 hsighxx20 THYDSSGL 8 THYDSSGLDAFDI 13 3 −4 1.72D+10 43 hsihr9 DDSSGS 6 DDSSGSYYFDY 11 4 −10 1.07D+09 44 hsihv11 LSGGYYS 7 LSGGYYSDFDY 11 4 −13 2.68D+08 45 hs ej1f GDYSDSSDSYI 11 GDYSDSSDSYIDAFDV 16 3 3 1.10D+12 46 hsmvh51 GETYYYDSRGYA 12 GETYYYDSRGYAFDH 15 4 6 2.62D+05 47 hsmvh517 PTRDSSGY 8 PTRDSSGYYVGY 12 4 −4 1.07D+09 48 hsmvh0406 GSFYYDSSGYPP 12 GSFYYDSSGYPPFDC 15 4 6 6.87D+10 49 hst14x14 GPYYYDSSGYYL 12 GPYYYDSSGYYLLDY 15 4 6 1.80D+16 50 hsvhig2 EEGYYDSSGYYSLGA 15 EEGYYDSSGYYSLGASDY 18 4 6 4.50D+15 51 hsvhia2 RPDSSGSRW 9 RPDSSGSRWYFDY 13 4 −7 6.71D+07 52 hsy14936 GYYDISGYYF 10 GYYDISGYYFDAFNI 15 3 −4 2.81D+14 53 hsy14934 DRGYDSSGYYGN 12 DRGYDSSGYYGNLDC 15 4 3 1.76D+13 54 hsy14935 DRGYDSIGYYGN 12 DRGYDSIGYYGNLDC 15 4 3 1.10D+12 55 hsz80519 AEDLTYYYDRSGWGVHGLL 19 AEDLTYYYDRSGWGVHGLLYYFDY 24 4 15 4.40D+12 56 hsz80429 LYPHYDSSGYYYV 13 LYPHYDSSGYYYVLDY 16 4 6 4.50D+15 57 hsz80461 DRVGYYDSSGYPPGSP 16 DRVGYYDSSGYPPGSPLDY 19 4 9 1.76D+13 Frequency of each AA type at each position in 57 Sequences having D3-22 segments Pos A C D E F G H I K L M N P Q R S T V W Y | X # 1 1 1 2 1 1 3 1 1 1 3 4 1 1 1 1 4 5 5 1 1 2 1 1 1 12 6 3 3 4 6 3 1 2 2 2 1 1 28 x 7 1 5 4 1 7 2 1 1 1 3 5 3 4 1 1 1 41 x 8 2 1 4 1 5 3 1 4 4 1 3 1 3 1 14 48 x 9 4 2 3 5 1 1 1 2 2 2 1 28 52 Y 10 1 4 2 1 1 1 1 4 1 40 56 Y 11 46 2 1 1 1 2 1 3 57 D 12 1 1 1 1 1 1 4 39 7 1 57 S 13 1 8 1 1 1 1 43 1 57 S 14 3 2 1 45 1 1 3 56 G 15 2 2 2 5 3 2 1 4 1 33 55 Y 16 2 1 1 1 2 3 1 1 1 6 3 1 1 1 24 49 x 17 3 1 1 1 5 2 1 4 6 6 2 7 2 1 1 3 46 x 18 8 1 1 2 2 2 4 3 1 3 27 19 2 1 1 1 3 4 1 13 20 2 1 2 1 1 1 1 9 21 1 1 1 3 22 1 1 2 23 1 1 2 24 1 1 25 1 1 Average Dseg = 11.9 Average DJ = 15.7 Median D = 12 12 Shortest 6 Longest 19 Median DJ = 15 15 Shortest 9 Longest 24

TABLE 13P Frequency of D-segments. “|” stands for a stop codon. D seg “0” % C % GLG “1” % C % GLG “2” % C % GLG 1-01 1 0.13 0 VQLERX 4 0.53 0.22 GTTGTX 5 0.66 0.34 YNWND 1-07 0 0 0 V|LELX 3 0.4 0.11 GITGTX 9 1.19 0.34 YNWNY 1-20 0 0 0 V|LERX 1 0.13 0.22 GITGTX 4 0.53 0.45 YNWND 1-26 4 0.53 0 V|WELLX 13 1.72 0.90 GIVGATX 36 4.76 0.78 YSGSYY 2-02 31 4.1 2.47 GYCSSTSCYT 4 0.53 0.22 RIL||YQLLYX 9 1.19 2.47 DIVVVPAAIX 2-08 5 0.66 0.56 GYCTNGVCYT 0 0 0 RILY|WCMLYX 3 0.4 0.56 DIVLMVYAIX 2-15 29 3.83 1.57 GYCSGGSCYS 2 0.26 0.11 RIL|WW|LLLX 7 0.92 1.57 DIVVVVAATX 2-21 16 2.11 0.67 AYCGGDCYS 0 0 0 SILWW|LLFX 7 0.92 0.67 HIVVVTAIX 3-03 32 4.23 2.80 YYDFWSGYYT 7 0.92 0.90 VLRFLEWLLYX 27 3.57 1.12 ITIFGVVIIX 3-09 13 1.72 1.35 YYDILTGYYN 5 0.66 0.78 VLRYFDWLL|X 0 0 0 ITIF|LVIIX 3-10 42 5.55 4.26 YYYGSGSYYN 13 1.72 0.89 VLLWFGELL|X 11 1.45 2.91 ITMVRGVIIX 3-16 18 2.38 0.67 YYDYVWGSYRYT 8 1.06 0 VL|LRLGELSLYX 5 0.66 0.34 IMITFGGVIVIX 3-22 57 7.53 3.36 YYYDSSGYYY 1 0.13 0.11 VLL|||WLLLX 6 0.79 0.34 ITMIVVVITX 4-04 5 0.66 0.28 DYSNY 2 0.26 0 |LQ|LX 2 0.26 0.06 TTVTX 4-17 29 3.83 1.45 DYGDY 0 0 0 |LR|LX 20 2.64 0.90 TTVTX 4-23 10 1.32 0.56 DYGGNS 1 0.13 0 |LRW|LX 4 0.53 0.56 TTVVTX 5-05 3 0.4 0.06 WIQLWLX 10 1.32 0.39 VDTAMVX 31 4.1 0.73 GYSYGY 5-12 0 0 0 WI|WLRLX 8 1.06 0.45 VDIVATIX 14 1.85 1.12 GYSGYDY 5-24 11 1.45 0 |RWLQLX 5 0.66 0.34 VEMATIX 13 1.72 0.44 RDGYNY 6-06 11 1.45 0.78 SIAARX 9 1.19 0.48 EYSSSS 1 0.13 0.11 V|QLVX 6-13 19 2.51 1.01 GIAAAGX 35 4.62 2.13 GYSSSWY 2 0.26 0.31 V|QQLVX 6-19 14 1.85 2.12 GIAVAGX 48 6.34 2.02 GYSSGWY 4 0.53 0.56 V|QWLVX D7: 7-27 1 0.13 0 |LGX 2 0.26 0.68 LTGX 2 0.26 0.22 NWG Total = 757

TABLE 14P Possible library components. Component L f D2_2-02_Phz0 xxxYCSSTSCxxx 13, 31, D3_3-16_Phz0 xxxxYVWGSYxxx 13, 18, D5_5-12_Phz2 xxxxxxxSGYxxx 13, 14, D3_3-09_Phz0 xxxYDILTGYYxx 13, 13, D2_2-02_Phz2 xxxVVVPAAxxxx 13, 9, D3_3-22_Phz0 xxxYYDSSGYxx 12, 57, D3_3-03_Phz0 xxxDFWSGxxxx 12, 32, D3_3-03_Phz2 xxxTIFGVxxxx 12, 27, D5_5-12_Phz1 xxxxIVATxxxx 12,  8, D3_3-10_Phz0 xxxYGSGSYYx 11, 42, ! could add one x at either end D5_5-05_Phz2 xxxxYSYGxxx 11, 31, D2_2-15_Phz0 xxxCSGxxCYx 11, 29, D6_6-13_Phz0 xxxxAAAGxxx 11, 19, D4_4-23_Phz0 xGxxxGGNxxx 11, 10, D1_1-26_Phz2 xxxSGSYxxx 10, 35, D6_6-13_Phz1 xxxSSSWxxx 10, 35, D4_4-17_Phz2 xxxxTTVTTx 10, 20, D2_2-21_Phz0 xxxC(SG)GDxCx 10, 16, D6_6-19_Phz0 xxx(IV)AVAGxx 10, 14, D3_3-10_Phz1 xxLWFGELxx 10, 13, D5_5-24_Phz0 GxxWLxxxxF 10, 11, D5_5-05_Phz1 xxxDTxMVxx 10, 10, D3_3-16_Phz1 xxxxxGExxx 10,  8, D6_6-19_Phz1 xxxxSGWxx  9, 48, D5_5-24_Phz2 xxxxGYNxx  9, 13, D3_3-10_Phz2 xxxVRGVxx  9, 11, D6_6-06_Phz0 xxxIAAxxx  9, 11, D1_1-07_Phz2 xxYxWNxxx  9,  9, D4_4-17_Phz0 xxxYGDxx  8, 29, D1_1-26_Phz1 xxVGATxx  8, 13, D6_6-06_Phz1 xxxXSSSx  8,  9,

TABLE 15P Lengths of CDRs: 1095 actual VH domains and 51 VH GLGs. Length 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 CDR1 0 0 10 0 1 820 38 175 1 1 5 1 11 0 23 1 7 0 GLG 0 0 0 0 0 38 3 10 0 0 . . . CDR2 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 464 579 GLG 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 17 28 CDR3 0 0 0 4 2 8 6 28 40 65  77 90 117 117 88 105 86 81 Length 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 (33 or more) CDR2 9 31 1 3 3 1 0 0 0 0 2 0 0 . . . GLG 1 4 0 0 . . . CDR3 45 36 36 16  16 8 8 2 3 0 2 1 0 0 1 5

TABLE 16P Library of HC CDR3 Bomponent Fraction of Length #X Complexity library Adjusted 1: YYCA21111YFDYWG. 8 4 2.6E5 .10 (0-8) .02 (2 = KR) 2: YYCA2111111YFDYWG. 10 6 9.4E7 .14 (9-10) .14 (2 = KR) 3: YYCA211111111YFDYTG. 12 8 3.4E10 .25 (11 + 12 + 13/2) .25 (2 = KR) 4: YYCAR111S2S3111YFDYWG. 14 6 1.9E8 .13 (14 + 13/2) .14 (2 = SG 3 = YW) 5: YYCA2111CSG11CY1YFDYWG. 15 6 9.4E7 .13 (15 + 16/2) .14 (2 = KR) 6: YYCA211S1TIFG11111YFDYWG. 17 8 1.7E10 .11 (17 + 16/2) .12 (2 = KR) 7: YYCAR111YY2S33YY111YFDYWG. 18 6 3.8E8 .04 (18) .08 (2 = D|G; 3 = S|G) 8: YYCAR1111YC2231CY111YFDYWG. 19 8 2.0E11 .10 (19 on) .11 (2 = S|G; 3 = T|D|G) Allowed lengths: 8, 10, 12, 14, 15, 17, 18, & 19

TABLE 17P vgDNA encoding the CDR3 elements of the library CDR3 library components (Ctop25) 5′-gctctggtcaa C|TTA|AGg|gct|gag|g-3′ (CtprmA) 5′-gctctggtcaa C|TTA|AGg|gct|gag|gac-                AflII... |acc|gct|gtc|tac|tac|tgc|gcc-3′ (CBprmB) [RC] 5′-|tac|ttc|gat|tac|ttg|ggc|caa|GGT|ACC|ctG|GTC|ACC|tcgctccacc-3′                                           BstEII... (CBot25) [RC] 5′-|GGT|ACC|ctG|GTC|ACC|tcgctccacc-3′ N.B. [RC] means the the actual oligonucleotide is the reverse complement of the one shown. N.B. The 20 bases at 3′ end of CtprmA are identical to the most 5′ 20 bases of each of the vgDNA molecules. N.B. Ctop25 is identical to the most 5′ 25 bases of CtprmA. N.B. The 23 most 3′ bases of CBprmB are the reverse complement of the most 3′ 23 bases of each of the vgDNA molecules. N.B. CBot25 is identical to the 25 bases at the 5′ end of CBprmB. (C1t08) 5′-cc|gct|gtc|tac|tac|tgc|gcc|- <2>|<1>|<1>|<1>|<1>- |tac|ttc|gat|tac|ttg|ggc|caa|GG-3′ 2 = KR, 1 = 0.27Y + 0.27G + 0.027{ADEFHIKLMNPQRSTVW} no C (C2t10) 5′-cc|gct|gtc|tac|tac|tgc|gcc|- <2>|<1>|<1>|<1>|<1>|<1>|<1>|- tac|ttc|gat|tac|ttg|ggc|caa|GG-3′ 2 = KR, 1 = 0.27Y + 0.27G + 0.027{ADEFHIKLMNPQRSTVW} no C (C3t12) 5′-cc|gct|gtc|tac|tac|tgc|gcc|- <2>|<1>|<1>|<1>|<1>|<1>|<1>|<1>|<1>|- tac|ttc|gat|tac|ttg|ggc|caa|GG-3′ 2 = KR, 1 = 0.27Y + 0.27G + 0.027{ADEFHIKLMNPQRSTVW} no C (C4t14) 5′-cc|gct|gtc|tac|tac|tgc|gcc|cgt|- |<1>|<1>|<1>|tct|<2>|tct|<3>|<1>|<1>|<1>|- tac|ttc|gat|tac|ttg|ggc|caa|GG-3′ 2 = SG, 1 = 0.27Y + 0.27G + 0.027{ADEFHIKLMNPQRSTVW} no C, 3 = YW (C5t15) 5′-cc|gct|gtc|tac|tac|tgc|gcc|- <2>|<1>|<1>|<1>|tgc|tct|ggt|<1>|<1>|tgc|tat|<1>|- tac|ttc|gat|tac|ttg|ggc|caa|GG-3′ 2 = KR, 1 = 0.27Y + 0.27G + 0.027{ADEFHIKLMNPQRSTVW} no C (C6t17) 5′-cc|gct|gtc|tac|tac|tgc|gcc|- <2>|<1>|<1>|tct|<1>|act|atc|ttc|ggt|<1>|<1>|<1>|<1>|<1>|- tac|ttc|gat|tac|ttg|ggc|caa|GG-3′ 2 = KR, 1 = 0.27Y + 0.27G + 0.027{ADEFHIKLMNPQRSTVW} no C (C7t18) 5′-cc|gct|gtc|tac|tac|tgc|gcc|cgt|- |<1>|<1>|<1>|tat|tac|<2>|tct|<3>|<3>|tac|tat|<1>|<1>|<1>|- tac|ttc|gat|tac|ttg|ggc|caa|GG-3′ 2 = DG, 1 = 0.27Y + 0.27G + 0.027{ADEFHIKLMNPQRSTVW} no C, 3 = SG (c8t19) 5′-cc|gct|gtc|tac|tac|tgc|gcc|cgt|- |<1>|<1>|<1>|<1>|tat|tgc|<2>|<2>|<3>|<1>|tgc|tat|<1>|<1>|<1>|- tac|ttc|gat|tac|ttg|ggc|caa|GG-3′ 2 = SG, 1 = 0.27Y + 0.27G + 0.027{ADEFHIKLMNPQRSTVW} no C, 3 = TDG

TABLE 19 Names of 1398 GeneBank entries examined haj10335 hsa006167 hsa234193 hsa234294 hsa239370 hsa241345 hsa244970 hs201e3 hsa006169 hsa234194 hsa234296 hsa239371 hsa241346 hsa244971 hs201g1 hsa006171 hsa234196 hsa234298 hsa239372 hsa241347 hsa244972 hs201m2 hsa006173 hsa234197 hsa235649 hsa239373 hsa241348 hsa244973 hs202e2 hsa131921 hsa234199 hsa235658 hsa239375 hsa241349 hsa244974 hs202g3 hsa132847 hsa234202 hsa235662 hsa239376 hsa241350 hsa244975 hs202g9 hsa132849 hsa234203 hsa235664 hsa239377 hsa241351 hsa244976 hs202m3 hsa132850 hsa234205 hsa235665 hsa239378 hsa241353 hsa244977 hs203e1 hsa132851 hsa234206 hsa235667 hsa239379 hsa241354 hsa244978 hs203g1 hsa132852 hsa234207 hsa235671 hsa239380 hsa241355 hsa244979 hs203m5 hsa224746 hsa234208 hsa235675 hsa239381 hsa241356 hsa244980 hs204e1 hsa225092 hsa234209 hsa235677 hsa239382 hsa241357 hsa244981 hs204g1 hsa225093 hsa234211 hsa238036 hsa239383 hsa241420 hsa244982 hs3d6hcv hsa230634 hsa234212 hsa238037 hsa239384 hsa241421 hsa244983 hs6d4xa7 hsa230635 hsa234214 hsa238038 hsa239385 hsa242555 hsa244984 hs6d4xb7 hsa230636 hsa234217 hsa238039 hsa239386 hsa242556 hsa244985 hs6d4xf1 hsa230637 hsa234221 hsa238040 hsa239387 hsa243108 hsa244986 hs6d4xf2 hsa230638 hsa234224 hsa238326 hsa239388 hsa243110 hsa244987 hs6d4xg3 hsa230639 hsa234227 hsa238327 hsa239390 hsa244928 hsa244988 hs6d4xh5 hsa230640 hsa234229 hsa238328 hsa239391 hsa244929 hsa244989 hs83x6b2 hsa230641 hsa234230 hsa239330 hsa240553 hsa244930 hsa244990 hs83x6b5 hsa230643 hsa234232 hsa239331 hsa240554 hsa244931 hsa244991 hs83x6c3 hsa230644 hsa234235 hsa239332 hsa240555 hsa244932 hsa244992 hs83x6c4 hsa230645 hsa234238 hsa239333 hsa240556 hsa244933 hsa244993 hs83x6c5 hsa230646 hsa234239 hsa239334 hsa240557 hsa244934 hsa244994 hs83x6d4 hsa230647 hsa234242 hsa239335 hsa240558 hsa244935 hsa244995 hs83x6f1 hsa230648 hsa234245 hsa239336 hsa240559 hsa244936 hsa244996 hs83x6f2 hsa230649 hsa234248 hsa239337 hsa240560 hsa244937 hsa244997 hs83x6f3 hsa230650 hsa234249 hsa239338 hsa240561 hsa244938 hsa244998 hs83x6f5 hsa230651 hsa234251 hsa239339 hsa240562 hsa244939 hsa244999 hs83x6h3 hsa230652 hsa234252 hsa239340 hsa240563 hsa244940 hsa245000 hs83x9a6 hsa230653 hsa234255 hsa239341 hsa240564 hsa244941 hsa245001 hs83x9b6 hsa230654 hsa234256 hsa239342 hsa240565 hsa244942 hsa245002 hs83x9b9 hsa230655 hsa234257 hsa239343 hsa240566 hsa244943 hsa245003 hs83x9c8 hsa230656 hsa234258 hsa239344 hsa240567 hsa244944 hsa245004 hs83x9d6 hsa230657 hsa234259 hsa239345 hsa240568 hsa244945 hsa245005 hs83x9d7 hsa230658 hsa234260 hsa239346 hsa240569 hsa244946 hsa245006 hs83x9e6 hsa234156 hsa234262 hsa239347 hsa240570 hsa244947 hsa245007 hs83x9e8 hsa234158 hsa234263 hsa239348 hsa240571 hsa244948 hsa245008 hs83x9e9 hsa234160 hsa234264 hsa239349 hsa240572 hsa244949 hsa245009 hs83x9f6 hsa234161 hsa234266 hsa239350 hsa240573 hsa244950 hsa245010 hs83x9g6 hsa234163 hsa234268 hsa239351 hsa240575 hsa244951 hsa245011 hs9d4x10 hsa234164 hsa234269 hsa239353 hsa240576 hsa244952 hsa245012 hs9d4x7 hsa234166 hsa234270 hsa239354 hsa240578 hsa244953 hsa245013 hs9d4x8 hsa234168 hsa234272 hsa239355 hsa240580 hsa244954 hsa245014 hs9d4x9 hsa234169 hsa234273 hsa239356 hsa240581 hsa244955 hsa245015 hs9d4xa6 hsa234171 hsa234274 hsa239357 hsa240582 hsa244956 hsa245016 hs9d4xa7 hsa234172 hsa234276 hsa239358 hsa240585 hsa244957 hsa245017 hs9d4xb6 hsa234175 hsa234277 hsa239359 hsa240586 hsa244958 hsa245018 hs9d4xc2 hsa234178 hsa234279 hsa239360 hsa240588 hsa244959 hsa245019 hs9d4xd6 hsa234180 hsa234281 hsa239361 hsa240589 hsa244960 hsa245020 hs9d4xe6 hsa234181 hsa234282 hsa239362 hsa240590 hsa244961 hsa245021 hs9d4xf3 hsa234183 hsa234283 hsa239363 hsa240592 hsa244962 hsa245022 hs9d4xh4 hsa234184 hsa234284 hsa239364 hsa240593 hsa244963 hsa245023 hs9d4xh5 hsa234186 hsa234286 hsa239365 hsa240594 hsa244965 hsa245024 hsa005975 hsa234187 hsa234287 hsa239366 hsa240595 hsa244966 hsa245025 hsa005977 hsa234189 hsa234288 hsa239367 hsa240599 hsa244967 hsa245026 hsa006161 hsa234190 hsa234290 hsa239368 hsa240604 hsa244968 hsa245027 hsa006165 hsa234191 hsa234291 hsa239369 hsa241344 hsa244969 hsa245028 hsa245029 hsa245225 hsa245319 hsa279536 hsasighc hsb8g3g01 hsfs11hc hsa245030 hsa245226 hsa245320 hsa279537 hsavh510 hsb8g3g03 hsfs9whc hsa245031 hsa245228 hsa245321 hsa279543 hsavh512 hsb8g3g05 hsgad2h hsa245032 hsa245229 hsa245322 hsa279544 hsavh513 hsb8g3g10 hsgvh0117 hsa245033 hsa245230 hsa245323 hsa279545 hsavh514 hsb8g3h01 hsgvh0118 hsa245034 hsa245231 hsa245325 hsa279552 hsavh515 hsb8g4c02 hsgvh0119 hsa245035 hsa245232 hsa245326 hsa389169 hsavh516 hsb8g4e01 hsgvh0120 hsa245036 hsa245233 hsa245338 hsa389170 hsavh517 hsb8g4e05 hsgvh0121 hsa245037 hsa245234 hsa245342 hsa389171 hsavh519 hsb8g4f11 hsgvh0122 hsa245039 hsa245235 hsa245343 hsa389172 hsavh520 hsb8g4h09 hsgvh0123 hsa245040 hsa245236 hsa245345 hsa389173 hsavh523 hsb8g4h10 hsgvh0124 hsa245041 hsa245237 hsa245346 hsa389174 hsavh524 hsb8g5d10 hsgvh0201 hsa245042 hsa245238 hsa245347 hsa389175 hsavh526 hsb8g5h08 hsgvh0202 hsa245043 hsa245239 hsa245348 hsa389176 hsavh529 hsbel1 hsgvh0203 hsa245044 hsa245240 hsa245349 hsa389177 hsavh53 hsbel14 hsgvh0204 hsa245045 hsa245241 hsa245350 hsa389178 hsavh56 hsbel28 hsgvh0205 hsa245046 hsa245246 hsa245352 hsa389179 hsb3g4a07 hsbel29 hsgvh0206 hsa245047 hsa245251 hsa245353 hsa389180 hsb73g04n hsbel3 hsgvh0207 hsa245048 hsa245255 hsa245355 hsa389181 hsb74a08n hsbel34 hsgvh0208 hsa245049 hsa245258 hsa245356 hsa389182 hsb7g1a11 hsbel43 hsgvh0209 hsa245050 hsa245260 hsa245357 hsa389183 hsb7g2b01 hsbel45 hsgvh0210 hsa245051 hsa245261 hsa245358 hsa389184 hsb7g3a01 hsbel5 hsgvh0211 hsa245052 hsa245262 hsa245359 hsa389185 hsb7g3a05 hsbel54 hsgvh0213 hsa245053 hsa245263 hsa249378 hsa389186 hsb7g3a10 hsbel69 hsgvh0214 hsa245054 hsa245265 hsa249628 hsa389187 hsb7g3b02 hsbo1vhig hsgvh0215 hsa245055 hsa245266 hsa249629 hsa389188 hsb7g3b03 hsbo3vhig hsgvh0216 hsa245056 hsa245268 hsa249630 hsa389190 hsb7g3b05 hsbr1vhig hsgvh0217 hsa245057 hsa245272 hsa249631 hsa389191 hsb7g3c03 hsbradh3 hsgvh0218 hsa245058 hsa245273 hsa249632 hsa389192 hsb7g3c12 hscal4ghc hsgvh0219 hsa245059 hsa245275 hsa249633 hsa389193 hsb7g3d07 hsd4xd10 hsgvh0220 hsa245060 hsa245277 hsa249634 hsa389194 hsb7g3e01 hsd4xf21 hsgvh0221 hsa245061 hsa245278 hsa249635 hsa389195 hsb7g3f02 hsd4xg2 hsgvh0222 hsa245062 hsa245279 hsa249636 hsa389927 hsb7g3f10 hsd4xi10 hsgvh0223 hsa245063 hsa245280 hsa249637 hsa389929 hsb7g3g02 hsd4xi4 hsgvh0224 hsa245064 hsa245281 hsa271600 hsa6351 hsb7g3g04 hsd4xk9 hsgvh0302 hsa245065 hsa245282 hsa271601 hsa7321 hsb7g4a08 hsd4xl3 hsgvh0304 hsa245066 hsa245283 hsa271602 hsa7322 hsb7g4c05 hsd5hc hsgvh0306 hsa245067 hsa245284 hsa271603 hsa7323 hsb7g4d09 hsdo1vhig hsgvh0307 hsa245068 hsa245285 hsa271604 hsa7325 hsb7g4f08 hseliepa1 hsgvh0308 hsa245069 hsa245286 hsa279513 hsa7326 hsb7g4g07 hseliepa3 hsgvh0309 hsa245071 hsa245287 hsa279514 hsa7328 hsb7g5g03 hseliepa4 hsgvh0310 hsa245072 hsa245288 hsa279515 hsa7438 hsb8g1c04 hseliepb2 hsgvh0311 hsa245073 hsa245289 hsa279516 hsa7440 hsb8g1e04 hseliepd2 hsgvh0312 hsa245201 hsa245290 hsa279517 hsa7441 hsb8g1f03 hselilpb1 hsgvh0314 hsa245203 hsa245291 hsa279519 hsa7442 hsb8g1g04 hsevh51a1 hsgvh0315 hsa245204 hsa245292 hsa279520 hsa7443 hsb8g1h02 hsevh51b1 hsgvh0318 hsa245208 hsa245294 hsa279521 hsa7444 hsb8g2f09 hsevh52a1 hsgvh0320 hsa245209 hsa245298 hsa279522 hsaarma1 hsb8g2g08 hsevh52a2 hsgvh0321 hsa245210 hsa245299 hsa279523 hsabhiv8 hsb8g3b07 hsevh52a3 hsgvh0322 hsa245214 hsa245301 hsa279524 hsadeigvh hsb8g3c07 hsevh52a4 hsgvh0323 hsa245215 hsa245305 hsa279526 hsaj2768 hsb8g3c08 hsevh52a5 hsgvh0324 hsa245217 hsa245307 hsa279527 hsaj2769 hsb8g3c12 hsevh52b1 hsgvh0325 hsa245218 hsa245309 hsa279528 hsaj2771 hsb8g3d03 hsevh53a1 hsgvh0326 hsa245219 hsa245311 hsa279529 hsaj2772 hsb8g3d04 hsevh53a2 hsgvh0327 hsa245220 hsa245312 hsa279530 hsaj2773 hsb8g3d07 hsfog1h hsgvh0328 hsa245221 hsa245313 hsa279531 hsaj2776 hsb8g3d08 hsfog3h hsgvh0329 hsa245222 hsa245315 hsa279532 hsaj2777 hsb8g3e02 hsfogbh hsgvh0330 hsa245223 hsa245317 hsa279533 hsaj4083 hsb8g3e03 hsfom1h hsgvh0331 hsa245224 hsa245318 hsa279535 hsaj4899 hsb8g3f03 hsfs10hc hsgvh0332 hsgvh0333 hsigathc hsighxx10 hsigvhc26 hsld1117 hsmvh51 hst14x23 hsgvh0334 hsigdvrhc hsighxx11 hsigvhc27 hsld152 hsmvh510 hst14x24 hsgvh0335 hsigg1kh hsighxx12 hsigvhc28 hsld21 hsmvh511 hst14x25 hsgvh0336 hsigg1kl hsighxx14 hsigvhc29 hsld217 hsmvh512 hst14x3 hsgvh0419 hsigg1lh hsighxx16 hsigvhc30 hsld218 hsmvh515 hst14x6 hsgvh0420 hsigghc85 hsighxx18 hsigvhc31 hsld25 hsmvh516 hst14x7 hsgvh0421 hsigghcv3 hsighxx2 hsigvhc32 hsmad2h hsmvh517 hst14x8 hsgvh0422 hsigghevr hsighxx20 hsigvhc33 hsmbcl5h4 hsmvh53 hst14x9 hsgvh0423 hsiggvdj1 hsighxx21 hsigvhc35 hsmica1h hsmvh54 hst22x1 hsgvh0424 hsiggvdj2 hsighxx22 hsigvhc36 hsmica3h hsmvh55 hst22x11 hsgvh0428 hsiggvhb hsighxx23 hsigvhc37 hsmica4h hsmvh56 hst22x12 hsgvh0429 hsiggvhc hsighxx25 hsigvhc38 hsmica5h hsmvh57 hst22x13 hsgvh0430 hsigh10g1 hsighxx26 hsigvhc39 hsmica6h hsmvh58 hst22x14 hsgvh0517 hsigh10g2 hsighxx28 hsigvhc40 hsmica7h hsmvh59 hst22x15 hsgvh0519 hsigh10g3 hsighxx29 hsigvhc41 hsmt11ige hsnamembo hst22x18 hsgvh0522 hsigh10g4 hsighxx3 hsigvhc42 hsmt12ige hsnpb346e hst22x20 hsgvh0523 hsigh10g5 hsighxx30 hsigvhc43 hsmt13ige hsoak3h hst22x21 hsgvh0526 hsigh10g7 hsighxx31 hsigvhls hsmt14ige hsog31h hst22x22 hsgvh0527 hsigh10g8 hsighxx32 hsigvhttd hsmt15ige hspag1h hst22x23 hsgvh0531 hsigh10g9 hsighxx34 hsigvp151 hsmt16ige hsrael hst22x25 hsgvh511 hsigh13g1 hsighxx36 hsigvp152 hsmt17ige hsregah hst22x26 hsgvh512 hsigh13g7 hsighxx37 hsigvp153 hsmt21ige hsrfabh37 hst22x27 hsgvh513 hsigh14g1 hsighxx38 hsigvp154 hsmt22ige hsrighvja hst22x28 hsgvh515 hsigh14g2 hsighxx5 hsigvp155 hsmt23ige hsrighvjb hst22x30 hsgvh519 hsigh2f2 hsighxx6 hsigvp156 hsmt24ige hsrou10 hst22x9 hsgvh521 hsigh3135 hsighxx7 hsigvp157 hsmt25ige hsrou11 hsu24687 hsgvh526 hsigh35 hsighxx8 hsigvp158 hsmt26ige hsrou111 hsu24688 hsgvh530 hsigh44 hsighxx9 hsigvp251 hsmt27ige hsrou112 hsu24690 hsgvh533 hsigh4c2 hsigkrf hsigvp255 hsmutuiem hsrou119 hsu24691 hsgvh534 hsigh9e1 hsigmhavh hsigvp256 hsmvh0001 hsrou122 hsv52a512 hsgvh535 hsighadi2 hsigrhe15 hsigvp257 hsmvh0002 hsrou126 hsvdj10h hsgvh536 hsighadi3 hsigtgk1h hsigvp360 hsmvh0003 hsrou127 hsvdj12h hsgvh55 hsighcvr hsigtgk4h hsigvp363 hsmvh0004 hsrou129 hsvgcg1 hsh217e hsighcza hsigtgl9h hsigvp369 hsmvh0005 hsrou13 hsvgcm1 hsh241e hsighczb hsigvarh1 hsigvp39 hsmvh0006 hsrou131 hsvgcm1 hsh28e hsighczc hsigvhc hsihr8 hsmvh0007 hsrou18 hsvh1djh6 hsha3d1ig hsighczd hsigvhc01 hsihr9 hsmvh0009 hsrou219 hsvh3djh4 hshambh hsighczf hsigvhc02 hsihv1 hsmvh0010 hsrou221 hsvh4dj hshcmg42 hsighczg hsigvhc03 hsihv11 hsmvh0011 hsrou222 hsvh4djh6 hshcmg43 hsigheavy hsigvhc04 hsihv18 hsmvh0012 hsrou233 hsvh4r hshcmg44 hsighpat2 hsigvhc05 hsim9vch hsmvh0401 hsrt792hc hsvh52a43 hshcmg46 hsighpat3 hsigvhc06 hsimghc1 hsmvh0403 hsrt79hc hsvh52a55 hshcmt42 hsighpat4 hsigvhc07 hsimghc2 hsmvh0404 hssm1vhig hsvh5dj hshcmt47 hsighpat5 hsigvhc08 hsimghc3 hsmvh0405 hssp46a hsvh5djh5 hsig001vh hsighpat6 hsigvhc09 hsimghc4 hsmvh0406 hst14vh hsvh710p1 hsig030vh hsighpat7 hsigvhc10 hsimghc5 hsmvh0501 hst14x1 hsvheg7 hsig033vh hsighpat8 hsigvhc11 hsin42p5 hsmvh0502 hst14x10 hsvhfa2 hsig039vh hsighpat9 hsigvhc12 hsin51p7 hsmvh0503 hst14x11 hsvhfa7 hsig040vh hsighpt11 hsigvhc14 hsin51p8 hsmvh0504 hst14x12 hsvhfb5 hsig055vh hsighpt12 hsigvhc16 hsin78 hsmvh0505 hst14x13 hsvhfc2 hsig057vh hsighpta1 hsigvhc17 hsin87 hsmvh0506 hst14x14 hsvhfd7 hsig1059 hsighvb5 hsigvhc18 hsin89p2 hsmvh0507 hst14x15 hsvhfe5 hsig10610 hsighvca hsigvhc19 hsin92 hsmvh0508 hst14x16 hsvhfg9 hsig13g10 hsighvcb hsigvhc20 hsin98p1 hsmvh0509 hst14x17 hsvhgd8 hsig473 hsighvcc hsigvhc21 hsjac10h hsmvh0510 hst14x18 hsvhgd9 hsig7sa11 hsighvcd hsigvhc22 hsjhba1f hsmvh0511 hst14x19 hsvhgh7 hsigaehc hsighvce hsigvhc23 hsjhbr2f hsmvh0513 hst14x20 hsvhha10 hsigaf2h2 hsighvm hsigvhc24 hsjhej1f hsmvh0515 hst14x21 hsvhia2 hsigashc hsighxx1 hsigvhc25 hsld1110 hsmvh0529 hst14x22 hsvhia5 hsvhib12 hsvhp46 hsx98948 hsz74671 hsz80393 hsz80438 hsz80489 hsvhib6 hsvhp48 hsx98950 hsz74672 hsz80394 hsz80439 hsz80492 hsvhib8 hsvhp53 hsx98951 hsz74682 hsz80397 hsz80441 hsz80495 hsvhic1 hsvhp7 hsx98952 hsz74688 hsz80400 hsz80442 hsz80496 hsvhic10 hsvigd9 hsx98953 hsz74690 hsz80403 hsz80443 hsz80499 hsvhic11 hswad35vh hsx98954 hsz74693 hsz80406 hsz80445 hsz80500 hsvhic2 hswanembo hsx98955 hsz74695 hsz80407 hsz80458 hsz80502 hsvhic3 hswo1vhig hsx98956 hsz80363 hsz80409 hsz80459 hsz80504 hsvhid1 hsww1p10e hsx98958 hsz80364 hsz80411 hsz80460 hsz80507 hsvhid5 hsx98932 hsx98963 hsz80365 hsz80412 hsz80461 hsz80509 hsvhid7 hsx98933 hsy14934 hsz80367 hsz80414 hsz80462 hsz80512 hsvhid9 hsx98934 hsy14935 hsz80368 hsz80415 hsz80463 hsz80513 hsvhie4 hsx98935 hsy14936 hsz80372 hsz80416 hsz80465 hsz80517 hsvhif10 hsx98936 hsy14937 hsz80375 hsz80417 hsz80466 hsz80519 hsvhif3 hsx98938 hsy14938 hsz80377 hsz80418 hsz80473 hsz80520 hsvhif7 hsx98939 hsy14939 hsz80378 hsz80421 hsz80474 hsz80527 hsvhig2 hsx98940 hsy14940 hsz80383 hsz80422 hsz80475 hsz80534 hsvhp2 hsx98941 hsy14943 hsz80385 hsz80424 hsz80476 hsz80538 hsvhp29 hsx98943 hsy14945 hsz80386 hsz80426 hsz80477 hsz80544 hsvhp30 hsx98944 hsy18120 hsz80388 hsz80427 hsz80480 hsz80545 hsvhp32 hsx98945 hsz74663 hsz80390 hsz80429 hsz80482 hsvhp34 hsx98946 hsz74665 hsz80391 hsz80433 hsz80483 hsvhp4 hsx98947 hsz74668 hsz80392 hsz80436 hsz80487

TABLE 20P Human GLG CDR1 & CDR2 AA seqs CDR2 CDR1          1    1   1 Name 1234567 1234567890123456789 1-02 GYY--MH WINPNSGG--TNYAQKFQG 1-03 SYA--MH WINAGNGN--TKYSQKFQG 1-08 SYD--IN WMNPNSGN--TGYAQKFQG 1-18 SYG--IS WISAYNGN--TNYAQKLQG 1-24 ELS--MH GFDPEDGE--TIYAQKFQG 1-45 YRY--LH WITPFNGN--TNYAQKFQD 1-46 SYY--MH IINPSGGS--TSYAQKFQG 1-58 SSA--VQ WIVVGSGN--TNYAQKFQE 1-69 SYA--IS GIIPIFGT--ANYAQKFQG 1-e SYA--IS GIIPIFGT--ANYAQKFQG 1-f DYY--MH LVDPEDGE--TIYAEKFQG 2-05 TSGVGVG LIYWNDDK---RYSPSLKS 2-26 NARMGVS HIFSNDEK---SYSTSLKS 2-70 TSGMRVS RIDWDDDK---FYSTSLKT 3-07 SYW--MS NIKQDGSE--KYYVDSVKG 3-09 DYA--MH GISWNSGS--IGYADSVKG 3-11 DYY--MS YISSSGST--IYYADSVKG 3-13 SYD--MH AIGTAGD---TYYPGSVKG 3-15 NAW--MS RIKSKTDGGTTDYAAPVKG 3-20 DYG--MS GINWNGGS--TGYADSVKG 3-21 SYS--MN SISSSSSY--IYYADSVKG 3-23 SYA--MS AISGSGGS--TYYADSVKG 3-30 SYG--MH VISYDGSN--KYYADSVKG 3303 SYA--MH VISYDGSN--KYYADSVKG 3305 SYG--MH VISYDGSN--KYYADSVKG 3-33 SYG--MH VIWYDGSN--KYYADSVKG 3-43 DYT--MH LISWDGGS--TYYADSVKG 3-48 SYS--MN YISSSSST--IYYADSVKG 3-49 DYA--MS FIRSKAYGGTTEYTASVKG 3-53 SNY--MS VIYSGGS---TYYADSVKG 3-64 SYA--MH AISSNGGS--TYYANSVKG 3-66 SNY--MS VIYSGGS---TYYADSVKG 3-72 DHY--MD RTRNKANSYTTEYAASVKG 3-73 GSA--MH RIRSKANSYATAYAASVKG 3-74 SYW--MH RINSDGSS--TSYADSVKG 3-d SNE--MS SISGGS----TYYADSRKG 4-04 SSNW-WS EIYHSGS---TNYNPSLKS 4-28 SSNW-WG YIYYSGS---TYYNPSLKS 4301 SGGYYWS YIYYSGS---TYYNPSLKS 4302 SGGYSWS YIYHSGS---TYYNPSLKS 4304 SGDYYWS YIYYSGS---TYYNPSLKS 4-31 SGGYYWS YIYYSGS---TYYNPSLKS 4-34 GYY--WS EINHSGS---TNYNPSLKS 4-39 SSSYYWG SIYYSGS---TYYNPSLKS 4-59 SYY--WS YIYYSGS---TNYNPSLKS 4-61 SGSYYWS YIYYSGS---TNYNPSLKS 4-b SGYY-WG SIYHSGS---TYYNPSLKS 5-51 SYW--IG IIYPGDSD--TRYSPSFQG 5-a SYW--IS RIDPSDSY--TNYSPSFQG 6-1 SNSAAWN RTYYRSKWY-NDYAVSVKS 74.1 SYA--MN WINTNTGN--PTYAQGFTG A C D E F G H I K L M N P Q R S T V W Y — Consens. CDR1 of human GLGs  1 7 1 3 2 35 2 1 Sd x  2 2 6 1 1 4 1  7 29 Ysg x  3 11  3 1 10  2 1  6 1 5 11 YAGS x  4 1 2 1 2  7 38 —  5 1 2 1  1  5 41 —  6 6 1 28  4 12  Mwi  7 1 5 16  5  1 23 SHng CDR2 of human GLGs  1 3 2 1 5 1 2 3 1 7  4 6 7  9 X  2 1 46 1 2 1 I  3 4 1 1 2 2 8 3 12 1 1 1 15 ysn x  4 2 2 4 1 10   1 11 2 1 5 12 ysp x  5 1 8 2 1 6 2 4 8 1 17  1 sd x  6 3 7 2 26  3  8 2 Gsd x  7 4 1 17  1 2 24  1  1 SG x  8 1 3 3 3 10   9 4 1  2 15 —ns  9 2  3 46 — 10 1 3 47 — 11 2 4 5 1 1 35   3 T 12 1 2 2 1 3 2 1 11  2  3 1 22 Yn x 13 51 Y 14 31  11  1  6 1 1 An x 15 4 16  1 1 1 14  11 2 1 dpq x 16 1 11  1 38 Sk 17 13  15  1 22  Vlf 18 37 13 1 Kq 19 1 1 34 14 1 GS

TABLE 21P Tallies of Amino-acid frequencies in mature CDR1 and CDR2 A C D E F G H I K L M N P Q R S T V W Y | X Tally of 23 examples with length 14 1 8 2 13 2 3 15 3 2 3 2 1 14 1 5 4 2 2 11 5 3 5 7 1 1 13 1 6 1 4 3 12 2 1 7 3 1 1 2 1 5 10 8 6 1 1 2 1 6 4 2 9 1 5 1 3 1 4 7 1 10 1 8 3 1 2 1 4 1 2 11 1 1 1 1 2 1 16 12 1 2 1 1 1 1 1 1 14 13 4 2 17 14 4 1 5 4 5 4 Tally of 11 examples with length 12 1 4 7 2 1 4 4 2 3 7 4 4 1 1 1 5 2 1 5 1 9 1 6 2 1 3 2 3 7 3 1 3 1 3 8 1 3 2 1 2 2 9 1 1 9 10 1 10 11 11 12 2 1 7 1 Tally of 175 examples with length 7 1 2 1 1 2 1 3 2 153 10 2 3 2 1 87 1 10 1 5 61 2 2 3 3 26 1 54 1 5 1 2 76 3 1 2 4 6 1 1 6 1 2 1 11 1 145 5 5 2 13 2 2 3 6 2 140 6 1 1 1 13 159 7 2 1 67 1 10 88 5 1 Tally of 38 examples with length 6 1 2 34 2 2 1 2 1 8 4 22 3 3 26 9 4 1 1 29 7 5 38 6 10 3 22 3 Tally of 820 examples with length 5 A C D E F G H I K L M N P Q R S T V W Y Seen 1 8 81 10 151 4 8 5 3 100 4 15 364 55 8  4 SGNDT 15 x 2 7 5 12 24 1 30 1 1 5 26 1 1 23 2 681 Y 15 3 202 4 24 13 13 133 10 2 7 5 2 3 32 14 13 112 231 YAGW 17 x 4 6 172 2 7 409 3 16 205 MWI 8 5 8 6 1 1 49 241 2 79 1 3 367 56 2  4 SHNT x 14 CDR2 A C D E F G H I K L M N Tally of 31 examples with CDR2 of length 19  1 11 1 1  2  1 28  3  9 1  4  1  2 6  5 1 1  1 22 1  6 16 1  1  1  1  7  1 9 7  8 23 1  9  2 18 10  4  1  1 1  1 11  1  3  1 12  2 11 9  1 1  1  1 13 1  1 14 29 15 25 3 1 16  1 17  1 1 18 1 27 19 1 30 Tally of 579 (n > 50, bold; over 400, underscored) examples with length 17  1 44 1 1 2 11 81 5 69  1 14 6 41  2 7 522    1 10 17  1  3  3 1 22 5  7 6 51 25  1 76  4 39 2 8 6 16 64 9  3  2  3 15  5  3 194 6 1 70 6 44  6  4 1 55  6  3 1 75 4 45 326  1  6 43  7  8 24 5 226  3  3  3  4 24  8  4 2 57 37 5 22 4 18 18  2 2 161   9 56 11  2 63 157   1 3  3 10  1 14 2 13 30 23  6 29  2 3 110  11  1 2 7 5  1  4  3 12 405   2 18  1  6  2 13  7 323 22  7 4  1  4 14  2 5 6  3 123  1  4 15  1 1 188  2  1 22 3 16  1 13  1 1 332   3 2  1 17 11 1 565   Tally of 464 (over 50, bold; over 400, underscored)  1  5 13 184 8  1 7  1  2 15  6  2 6 429    3 4  3 1 13 13  4 10  5 154   4  1 12 2 6 199  2  1  3  5  5 20 1 1 18  4  9  6 13 8 439    1  7 20 2 14 2  4  2 26  8 13  2  4  8  1  2  9 10 4  1 10  1  8  1 245  10  6 2 2  2 11 14 3 1  1  8 408   12  4 13  4  2  1 13  2 2 14 2  2 441   15 18 413   3  5 16  1 1 31  2  2 P Q R S T V W Y X Tally of 31 examples with CDR2 of length 19  1 1 15  1  1 RF x  2  2 I  3 18  1  1  1 Rk  4 21  1 S  5 1 1  1  1  1 K x  6  3  1  6 1 A x  7  3  1 10 y x  8 1  5  1 G  9 1  1 1  7 1 G 10  1 21  1 T 11 26 T 12  2  1  2 x 13 29 Y 14  1  1 A 15 1  1 A 16 10 20 Sp 17 29 V 18 1 2 K 19 G Tally of 579 (n > 50, bold; over 400, underscored) examples with length 17  1  1 4 34 30 19 118 66 31 VGIW x  2  3  8 10 I  3 8 262  19 1 46 46 SNI x  4 178  23 6 50 11 8 16 120  PYG x  5 4 8 133   9 7 1 27 DSGN x  6 1 63  8 1  2 GDS x  7 2 11 245  14 6  1 SG x  8  1 4 11 106  90 2 1 32 NST X  9 11 5 13  4 242  8 TKIA x 10 3 52 20 10 1 1 259  YNR x 11  5 551   Y 12  3 1 89  8 44 A 13 66 138 3 1  3 DQP x 14 2 7 421    1 2  2 SK x 15  1 357  2 1 VF 16  1 199 21  4 KQ x 17  1 1 G Tally of 464 (over 50, bold; over 400, underscored)  1 3 26 65  9 14 105  EYSL x  2  1  2 19 I  3 1 12 1  250  YN x  4  4 5 2 19 28 15 165  YH x  5  1 22 365  16 1  1 S x  6 1  1 1 G  7  1 12 357  20 1  2 1 S x  8  4 3  6 420   1 T  9 13  9  3 1 1 157  NY x 10 1  7 444   Y 11  4 21  2  2 N 12 418   14  7 1 P 13  6 452    1  1 S 14 1 18 L 15 11 10  1  2 1 K 16 3 419    5 S

TABLE 22P Tally of VH types 1-02 16 1-03 16 1-08 13 1-18 27 1-24 5 1-45 0 1-46 14 1-58 1 1-69 37 1-e 16 1-f 1 2-05 13 2-26 1 2-70 2 3-07 33 3-09 13 3-11 15 3-13 4 3-15 10 3-20 4 3-21 25 3-23 85 3-30 55 3303 59 3305 0 3-33 42 3-43 1 3-48 24 3-49 11 3-53 12 3-64 4 3-66 4 3-72 3 3-73 3 3-74 12 3-d 0 4-04 29 4-28 3 4301 46 4302 7 4304 37 4-31 0 4-34 184 4-39 65 4-59 45 4-61 9 4-b 11 5-51 55 5-a 13 6-1 7  74.1 3

TABLE 23P Oligonucleotides used to variegate CDR1 and CDR2 of human HC (name) 5′-....DNA sequence....-3′ everything to right of an exclamation point is commentary [RC] means “reverse complement” of sequence shown If last non-comment and non-blank character is “-”, then continue on next line. Ignore case, “a” = “A”, “c” = “C”, etc. Ignore “|” and blanks. <number> means incorporate trinucleotide mixtue of given number. CDR1 (ON-R1V1vg) 5′-ct|TCC|GGA|ttc|act|ttc|tct|- <1>|tac|<1>|atg|<1>|-! CDR1 of length 5, ON = 55 bases tgg|gtt|cgC|CAa|gct|ccT|GG-3′ <1> = ADEFGHIKLMNPQRSTVWY no C (ON-R1top) 5′-cctactgtct |TCC|GGA|ttc|act|ttc|tct-3′ (ON-R1bot) [RC] 5′-tgg|gtt|cgC|CAa|gct|ccT|GG ttgctcactc-3′ (ON-R1V2vg) 5′-ct|TCC|GGA|ttc|act|ttc|tct|- <6>|<7>|<7>|tac|tac|tgg|<7>|-! CDR1 of length 7, ON = 61 bases tgg|gtt|cgC|CAa|gct|ccT|GG-3′ <6> = ST, 1:1 <7> = 0.2025(SG) + 0.035(ADEFHIKLMNPQRTVWY) no C (ON-R1V3vg) 5′-ct|TCC|GGA|ttc|act|ttc|tct|- |atc|agc|ggt|ggt|tct|atc|tcc|<1>|<1>|<1>|tac|tac|tgg|<1>|-! CDR1, L = 14 tgg|gtt|cgC|CAa|gct|ccT|GG-3′ ! ON = 82 bases CDR2 (ON-R2V1vg) 5′-ggt|ttg|gag|tgg|gtt|tct|- <2>|atc|<2>|<3>|tct|ggt|ggc|<1>|act|<1>|- tat|gct|gac|tcc|gtt|aaa|gg-3′ ! ON = 68 bases, CDR2 = 17 AA (ON-R2top) 5′-ct|tgg|gtt|cgC|CAa|gct|ccT|GGt|aaa|ggt|ttg|gag|tgg|gtt|tct-3′ (ON-R2bot) [RC] 5′-tat|gct|gac|tcc|gtt|aaa|ggt|- cgc|ttc|act|atc|TCT|AGA|ttcctgtcac-3′ ! XbaI plus 10 bases of scab (ON-R2V2vg) 5′-ggt|ttg|gag|tgg|gtt|tct|- <1>|atc|<4>|<1>|<1>|ggt|<5>|<1>|<1>|<1>|- tat|gct|gac|tcc|gtt|aaa|gg-3′ ! ON = 68 bases, CDR2 = 17 AA <4> = DINSWY, equimolar <5> = SGDN, equimolar (ON-R2V3vg) 5′-ggt|ttg|gag|tgg|gtt|tct|- <1>|atc|<4>|<1>|<1>|ggt|<5>|<1>|<1>|- tat|aac|cct|tcc|ctt|aag|gg-3′ ! ON = 65 bases, CDR2 = 16 AA (ON-R2bo3) [RC] 5′-tat|aac|cct|tcc|ctt|aag|ggt|- cgc|ttc|act|atc|TCT|AGA|ttcctgtcac-3′ ! XbaI plus 10 bases of scab (ON-R2V4vg) 5′-ggt|ttg|gag|tgg|gtt|tct|- <1>|atc|<8>|agt|<1>|<1>|<1>|ggt|ggt|act|act|<1> tat|gcc|gct|tcc|gtt|aag|gg-3′ ! ON = 74 bases, CDR2 = 19 AA (ON-R2bo4) [RC] 5′-tat|gcc|gct|tcc|gtt|aag|ggt|- cgc|ttc|act|atc|TCT|AGA|ttcctgtcac-3′ ! XbaI plus 10 bases of scab

TABLE 25P Lengths of CDRs in 285 human kappa chains 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 CDR1 0 0 0 0 0 0 0 0 0 0 0 154 73 3 0 0 28 27 0 0 CDR2 0 0 0 0 0 0 0 285 0 0 0 0 0 0 0 0 0 0 0 0 CDR3 0 5 0 0 1 0 3 2 28 166 63 12 1 1 0 0 0 0 0 1

TABLE 26P Tally of kappa types: V and J V genes: O12 59 O2 0 O18 0 O8 0 A20 0 A30 0 L14 0 L1 2 L15 0 L4 2 L18 0 L5 4 L19 0 L8 4 L23 0 L9 1 L24 0 L11 4 L12 8 O11 10 O1 0 A17 5 A1 0 A18 3 A2 0 A19 13 A3 0 A23 4 A27 79 A11 26 L2 28 L16 0 L6 11 L20 0 L25 0 B3 22 B2 0 A26 0 A10 0 A14 0 JH# 1  2 3  4 5 tally 105 64 29 78 9

TABLE 27P Names of Kappa chains analyzed   AB022651 AB022653 AB022654 AB022656 AF007572 AF021036 AF103499 AF103500 AF103527 AF103873 AF107244 AF107245 AF107246 AF107247 AF115361 AF165099 AF165101 AF165103 AF165108 AF165110 AF165111 AF184763 AF184767 hsa004955 hsa004956 hsa011133 HSA241367 HSA241375 HSA388639 HSA388640 HSA388641 HSA388642 HSA388643 HSA388644 HSA388645 HSA388646 HSA388647 HSA388648 HSA388650 HSA388651 HSA388652 HSA388653 HSA388654 HSA388655 HSA388656 HSA388657 hsew1vk hsew3vk hsew4vk hsigdpk13 hsigg1kl HSIGGVKA hsigk123 hsigk319 hsigklc14 hsigklc28 hsigklc5 hsigklg31 hsigklv01 hsigklv02 hsigklv03 hsigklv04 hsigklv05 hsigklv06 hsigklv07 hsigklv09 hsigklv10 hsigklv12 hsigklv13 hsigklv14 hsigklv15 hsigklv16 hsigklv17 hsigklv18 hsigklv19 hsigklv20 hsigklv21 hsigklv22 hsigklv23 hsigklv24 hsigklv25 hsigklv27 hsigklv28 hsigklv29 hsigklv31 hsigklv32 hsigklv33 hsigklv34 hsigklv35 hsigklv36 hsigklv37 hsigklv38 hsigklv39 hsigklv40 hsigklv41 hsigklv42 hsigklv43 hsigklv44 hsigklv45 hsigklv46 hsigklv49 hsigklv50 hsigklv51 hsigklv52 hsigklv53 hsigklv54a hsigklv56 hsigklv57 hsigklv58 hsigklv59 hsigklv60 hsigklv61 hsigklv62 hsigklv63 hsigklv65 hsigklv66 hsigklv68 hsigklv69 hsigklv71 hsigkvba hsigkvbb hsigkvbc hsigkvbd hsigkvbe hsigkvbf hsigkvc01 hsigkvc03 hsigkvc06 hsigkvc11 hsigkvc12 hsigkvc20 hsigkvc23 hsigkvc27 hsigkvc29 hsigrklc hsikcvjp1 hsikcvjp2 hsikcvjp3 hsikcvjp6 hsikcvjp7 hsld110vl hsld117vl hsld128vl hsld140vl hsld152vl hsld184vl hsld198vl hsld24vl hsmbcl1k1 hsmbcl1k2 hsmbcl2k2 hsmbcl5k4 hssl0avl hss17bvl hss1a15vl HSU44792 HSU44794 HSU94422 hsz84852 hsz84853 humigk1dm humigk3am humigk3bm humigk3cm humigkacoa humigkacob humigkacoc humigkacoe humigkacof humigkb1aa humigkb1ab humigkb1ac humigkvra humigkvrb humigkvrc humigkvrd humigkvre humigkvrg humigkvrh humigkvri humigkx humigky1 humigky2 humigky4 humigky5 humigky6 humigl3ac humikc humikca humikcad humikcaf humikcag humikcah humikcai humikcaj humikcal humikcam humikcan humikcas humikcau humikcav humikcaw humikcax humikcay humikcaz humikcb humikcba humikcbb humikcbc humikcbd humikcbe humikcbf humikcbg humikcbh humikcbi humikcbj humikcbl humikcbm humikcbn humikcbo humikcbp humikcbq humikcbs humikcbt humikcbu humikcbv humikcbw humikcbx humikcbz humikcc humikcca humikccb humikccc humikccd humikcce humikccf humikccg humikcch humikcci humi kccj humikcck humikcco humikccp humikccq humikccr humikccs humikcct humikccu humikccv humikccw humikcd humikcf humikcg humikch humikci humikck humikcm humikcn humikco humikcp humikcq humikcr humikcs humikct humikcu humikcv humikcva humikcvb humikcvc humikcvd humikcve humikcvf humikcvg humikcvh humikcvi humikcvj humikcw humikcx humikcy humikcz S46248 582746 S82747 SU96396 SU96397

TABLE 28P AA types seen in 154 kappa sequences having CDR1 of length 11 Tally A C D E F G H I K L M N P Q R S T V W Y 1 11 143 R 2 148 1 2 2 1 A 3 152 2 S 4 1 3 3 147 Q 5 12 1 27 7 3 99 4 1 S 6 1 81 1 71 V 7 2 4 18 5 1 2 9 12 97 3 1 S 8 3 5 1 2 1 31 1 10 87 12 1 S 9 2 7 10 1 6 29 1 8 13 77 Y 10 2 150 1 1 L 11 96 4 2 46 2 1 3 A

TABLE 30P Synthetic Kappa light chain gene 1 GAGGACCATt GGGCCCC                 ctccgagact Scab...... EcoO109I            ApaI. 28 CTCGAG    cgca XhoI.. 38 acgcaatTAA TGTgagttag ctcactcatt aggcacccca ggcTTTACAc tttatgcttc        ..-35..         Plac                    ..-10. 98 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tc 140 acacagga aacagctatgac 160 catgatta cgCCAAGCTT TGGagccttt tttttggaga ttttcaac            PflMI.......              Hind3. M13 III signal sequence (AA seq)--------------------------->  1   2   3   4   5   6   7   8   9  10  11  12  13  14  15  M   K   K   L   L   F   A   I   P   L   V   V   P   F   Y 206 gtg aag aag ctc cta ttt gct atc ccg ctt gtc gtt ccg ttt tac --Signal-->  FR1------------------------------------------->  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30   S   H   S   A   Q   S   V   L   T   Q   S   P   G   T   L 251 |agc|cat|aGT|GCA|Caa|tcc|gtc|ctt|act|caa|tct|cct|ggc|act|ctt|           ApaLI... ----- FR1 ------------------------------------->| CDR1------>  31  32  33  34  35  36  37  38  39  40  41  42  43  44  45   S   L   S   P   G   E   R   A   T   L   S   C   R   A   S |tcG|CTA|AGC|CCG|GGt|gaa|cgt|gct|acC|TTA|AGt|tgc|cgt|gct|tcc|    EspI.....                       AflII...            XmaI.... For CDR1: <1> ADEFGHIKLMNPQRSTVWY equimolar <2> S(0.2) ADEFGHIKLMNPQRTVWY (0.044 each) <3> Y(0.2) ADEFGHIKLMNPQRSTVW (0.044 each) In a preferred embodiment, we omit codon 52 in vgDNA for CDR1. ------- CDR1 --------------------->|--- FR2 ---------------->     <1>     <2> <2> xxx <3> 46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  Q   S   V   S   S   S   Y   L   A   W   Y   Q   Q   K   P |cag|tct|gtt|tcc|tct|tct|tat|ctt|gct|tgg|tat|caa|cag|aaA|CCT|                                                        SexAI... For CDR2: <1> ADEFGHIKLMNPQRSTVWY equimolar <2> S(0.2) ADEFGHIKLMNPQRTVWY (0.044 each) <4> A(0.2) DEFGHIKLMNPQRSTVWY (0.044 each) ----- FR2 ------------------------->|------- CDR2 ---------->                                      <1>         <2>     <4>  61  62  63  64  65  66  67  68  69  70  71  72  73  74  75   G   Q   A   P   R   L   L   I   Y   G   A   S   S   R   A |GGT|caG|GCG|CCg|cgt|tta|ctt|att|tat|ggt|gct|tct|tcc|cgc|gct| SexAI.... KasI.... (CDR1 installed as AflII-(SexAI or KasI) cassette.) CDR2-->|--- FR3 ----------------------------------------------->  <1>  76  77  78  79  80  81  82  83  84  85  86  87  88  89  90   T   G   I   P   D   R   F   S   G   S   G   S   G   T   D |act|gGG|ATC|CCG|GAC|CGt|ttc|tct|ggc|tct|ggt|tca|ggt|act|gac|       BamHI...              RsrII..... (CDR2 installed as (SexAI or KasI) to (BamHI or RsrII) cassette.) ------ FR3 ------------------------------------------------->  91  92  93  94  95  96  97  98  99 100 101 102 103 104 105   F   T   L   T   I   S   R   L   E   P   E   D   F   A   V 477 |ttt|acc|ctt|act|att|TCT|AGA|ttg|gaa|cct|gaa|gac|ttc|gct|gtt|                      XbaI... ----------->|----CDR3-------------------------->|-----FR4--->                      <3> <1> <1> <1>     <1> 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120   Y   Y   C   Q   Q   Y   G   S   S   P   E   T   F   G   Q |tat|tat|tgC|CAa|cag|taT|GGt|tct|tct|cct|gaa|act|ttc|ggt|caa|            BstXI........... -----FR4------------------->|  <------- Ckappa ------------ 121 122 123 124 125 126 127    128 129 130 131 132 133 134    G   T   K   V   E   I   K     R   T   V   A   A   P   S 510 |ggt|aCC|AAG|Gtt|gaa|atc|aag| |CGT|ACG|gtt|gcc|gct|cct|agt|       StyI....                 BsiWI.. (CDR3 installed as XbaI to (StyI or BsiWI) cassette.)  135 136 137 138 139 140 141 142 143 144 145 146 147 148 149   V   F   I   F   P   P   S   D   E   Q   L   K   S   G   T 552 |gtg|ttt|atc|ttt|cct|cct|tct|gac|gaa|CAA|TTG|aag|tca|ggt|act|                                      MfeI...  150 151 152 153 154 155 156 157 158 159 160 161 162 163 164   A   S   V   V   C   L   L   N   N   F   Y   P   R   E   A 597 |gct|tct|gtc|gta|tgt|ttg|ctc|aac|aat|ttc|tac|cCT|CGT|Gaa|gct|                                               BssSI...  165 166 167 168 169 170 171 172 173 174 175 176 177 178 179   K   V   Q   W   K   V   D   N   A   L   Q   S   G   N   S 642 |aaa|gtt|cag|tgg|aaa|gtc|gat|aAC|GCG|Ttg|cag|tcg|ggt|aac|agt|                               MluI....  180 181 182 183 184 185 186 187 188 189 190 191 192 193 194   Q   E   S   V   T   E   Q   D   S   K   D   S   T   Y   S 687 |caa|gaa|tcc|gtc|act|gaa|cag|gat|agt|aag|gac|tct|acc|tac|tct|  195 196 197 198 199 200 201 202 203 204 205 206 207 208 209   L   S   S   T   L   T   L   S   K   A   D   Y   E   K   H 732 |ttg|tcc|tct|act|ctt|act|tta|tca|aag|gct|gat|tat|gag|aag|cat|  210 211 212 213 214 215 216 217 218 219 220 221 222 223 224   K   V   Y   A   C   E   V   T   H   Q   G   L   S   S   P 777 |aag|gtc|tat|GCt|TGC|gaa|gtt|acc|cac|cag|ggt|ctG|AGC|TCc|cct|                                                SacI....  225 226 227 228 229 230 231 232 233 234   V   T   K   S   F   N   R   G   E   C   .   . 822 |gtt|acc|aaa|agt|ttc|aaC|CGT|GGt|gaa|tgc|taa|tag GGCGCGCC                        DsaI....                  AscI....                                                   BssHII 866 acgcatctctaa GCGGCCGC aacaggaggag              NotI....               EagI.. A27::JH1 with all CDRs replaced by stuffers. Each stuffer contains at least one stop codon and a restriction site that will be unique within the diversity vector.

TABLE 31P Tally of 285 CDR2s of length 7 in human kappa Tally A C D E F G H I K L M N P Q R S T V W Y 1 51 62 7 95 1 11 15 2 1 2 6 6 3 22 1 x 2 225 18 5 5 2 1 1 3 16 9 A 3 2 9 1 2 267 2 1 1 S 4 2 1 5 4 9 1 77 4 93 80 2 7 Sx 5 1 2 80 200 2 R 6 162 7 36 4 4 1 3 3 63 2 Ax 7 5 1 3 1 1 2 2 1 125 144 x

TABLE 32P Tally of 166 CDR3s of length 9 from human kappa. Tally A C D E F G H I K L M N P Q R S T V W Y 1 4 8 21 131 1 1 Q 2 1 9 2 1 153 Q 3 14 4 4 3 6 4 1 1 3 21 16 3 4 82 Yx 4 1 9 1 2 37 4 2 2 15 1 33 2 20 7 1 29 x 5 2 2 6 3 4 5 3 28 17 7 65 19 1 1 3 x 6 7 1 11 2 3 8 1 4 3 41 33 5 28 19 x 7 1 2 6 146 2 2 5 2 P 8 2 4 1 2 21 7 3 5 1 38 7 4 25 1 3 1 16 25 x 9 3 2 1 1 2 157 T

TABLE 33P lengths of CDRs in 93 human lambda chains 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18+ CDR1 0 0 0 0 0 0 0 0 0 0 0 23 7 15 46 0 0 0 2 CDR2 5 0 0 1 0 0 0 80 2 0 0 1 4 0 0 0 0 0 1 CDR3 0 0 0 0 0 0 0 0 1 16 28 27 6 1 0 4 6 4 0

TABLE 34P Tally of 46 CDR1s of length 14 from human lambda chains Tally A C D E F G H I K L M N P Q R S T V W Y 1 2 2 1 41 T 2 43 3 G 3 2 1 1 6 36 TGx 4 1 45 S 5 5 1 40 S 6 39 1 4 2 DNx 7 8 1 37 V 8 1 42 2 1 G 9 4 1 35 1 2 3 TGx 10 1 1 3 1 2 38 Yx 11 4 1 35 6 DNx 12 3 1 2 1 1 2 36 Yx 13 1 2 43 V 14 1 4 41 S

TABLE 35P Synthtic human lambda-chain gene 1 GAGGACCATt GGGCCCC   ttactccgtgac Scab...... EcoO109I            ApaI..         -----------FR1-------------------------------------------->          1   2   3   4   5   6   7   8   9  10  11  12  13  14  15  S   A   Q   S   A   L   T   Q   P   A   S   V   S   G   S   P   G 30 aGT|GCA|Caa|tcc|gct|ctc|act|cag|cct|GCT|AGC|gtt|tcc|gGG|TcA|CCt|GGT|  ApaLI...                           NheI...          BstEII...                                                           SexAI.... For CDR1, <1> = 0.27 T, 0.27 G, 0.027 {ADEFHIKLMNPQRSVWY} no C <2> = 0.27 D, 0.27 N, 0.027 {AEFGHIKLMPQRSTVWY} no C <3> = 0.36 Y, 0.0355{ADEFGHIKLMNPQRSTVW} no C                               T   G  <1>  S   S  <2>  V   G ------FR1------------------> |-----CDR1---------------------  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30   Q   S   I   T   I   S   C   T   G   T   S   S   D   V   G |caa|agt|atc|act|att|tct|TGT|ACA|ggt|act|tct|tct|gat|gtt|ggc|                          BsrGI.. a second vg scheme for CDR1 gives segments of length 11: G₂₃<2><4>L<4><4><4><3><4><4> where <4> = equimolar {ADEFGHIKLMNPQRSTVWY} no C <1> <3> <2> <3>  V   S = vg Scheme #1, length = 14 -----CDR1------------->|--------FR2------------------------- 31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  G   Y   N   Y   V   S   W   Y   Q   Q   H   P   G   K   A |ggt|tac|aat|tac|gtt|tct|tgg|tat|caa|caa|caC|CCG|GGc|aaG|GCG|                                            XmaI....    KasI.....                                            AvaI....                         <4> <4> <4> <2>  R   P   S --FR2-----------------> |------CDR2--------------->|-----FR3-- 46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  P   K   L   M   I   Y   E   V   S   N   R   P   S   G   V |CCg|aag|ttg|atg|atc|tac|gaa|gtt|tcc|aat|cgt|cct|tct|ggt|gtt| KasI.... -------FR3----------------------------------------------------  61  62  63  64  65  66  67  68  69  70  71  72  73  74  75   S   N   R   F   S   G   S   K   S   G   N   T   A   S   L |agc|aat|cgt|ttc|TCC|GGA|tct|aaa|tcc|ggt|aat|acc|gcA|AGC|TTa|                  BspEI..          |                HindIII.                       BsaBI........(blunt) -------FR3--------------------------------------------------->|  76  77  78  79  80  81  82  83  84  85  86  87  88  89  90   T   I   S   G   L   Q   A   E   D   E   A   D   Y   Y   C |act|atc|tct|ggt|CTG|CAG|gct|gaa|gac|gag|gct|gac|tac|tat|tgt|                  PstI... <5> = 0.36 S, 0.0355{ADEFGHIKLMNPQRTVWY} no C  <4> <5> <4> <2> <4>  S  <4> <4> <4> <4>  V -----CDR3---------------------------------->|---FR4---------  91  92  93  94  95  96  97  98  99 100 101 102 103 104 105   S   S   Y   T   S   S   S   T   L   V   V   F   G   G   G |tct|tct|tac|act|tct|tct|agt|acc|ctt|gtt|gtc|ttc|ggc|ggt|GGT|                                                          KpnI... -------FR4-------------->  106 107 108 109 110 111 112 113 114 115 116 117 118 119 120   T   K   L   T   V   L   G   Q   P   K   A   A   P   S   V 279 |ACC|aaa|ctt|act|gtc|ctc|gGT|CAA|CCT|aAG|Gct|gct|cct|tcc|gtt| KpnI...                      HincII..                                  Bsu36I...  121 122 123 124 125 126 127 128 129 130 131 132 133 134 135   T   L   F   P   P   S   S   E   E   L   Q   A   N   K   A 324 |act|ctc|ttc|cct|cct|agt|tct|GAA|GAG|Ctt|caa|gct|aac|aag|gct|                              SapI.....  136 137 138 139 140 141 142 143 144 145 146 147 148 149 150   T   L   V   C   L   I   S   D   F   Y   P   G   A   V   T 369 |act|ctt|gtt|tgc|tTG|ATC|Agt|gac|ttt|tat|cct|ggt|gct|gtt|act|                   BclI....  151 152 153 154 155 156 157 158 159 160 161 162 163 164 165   V   A   W   K   A   D   S   S   P   V   K   A   G   V   E 414 |gtc|gct|tgg|aaa|gcc|gat|tct|tct|cct|gtt|aaa|gct|ggt|gtt|GAG|                                                          BsmBI...  166 167 168 169 170 171 172 173 174 175 176 177 178 179 180   T   T   T   P   S   K   Q   S   N   N   K   Y   A   A   S 459 |ACG|acc|act|cct|tct|aaa|caa|tct|aac|aat|aag|tac|gct|gcG|AGC| BsmBI....                                              SacI....  181 182 183 184 185 186 187 188 189 190 191 192 193 194 195   S   Y   L   S   L   T   P   E   Q   W   K   S   H   K   S 504 |TCt|tat|ctt|tct|ctc|acc|cct|gaa|caa|tgg|aag|tct|cat|aaa|tcc| SacI...  196 197 198 199 200 201 202 203 204 205 206 207 208 209 210   Y   S   C   Q   V   T   H   E   G   S   T   V   E   K   T 549 |tat|tcc|tgt|caa|gtt|acT|CAT|GAa|ggt|tct|acc|gtt|gaa|aag|act|                        BspHI...  211 212 213 214 215 216 217 218 219   V   A   P   T   E   C   S   .   . 594 |gtt|gcc|cct|act|gag|tgt|tct|tag|tga|GGCGCGCC                                      AscI....                                       BssHII 629 aacgatgttc aag GCGGCCGC aacaggaggag                NotI.... Scab....... Lambda 14-7(A) 2a2 ::JH2::Clambda AA sequence tested

TABLE 36P Tally of 23 CDR1s of length 11 from human lambda chains Tally A C D E F G H I K L M N P Q R S T V W Y 1 1 6 10 6 x 2 1 1 21 G 3 15 1 7 DNx 4 2 1 1 3 7 1 8 X 5 7 16 L 6 11 1 2 8 1 X 7 1 1 1 2 2 1 14 1 X 8 1 10 1 1 1 2 7 X 9 2 6 15 Yx 10 11 1 11 X 11 3 7 9 2 2 X

TABLE 37P Tally of 80 CDR2s of length 7 from human lambda chains Tally A C D E F G H I K L M N P Q R S T V W Y 1 1 14 32 1 13 3 1 4 5 1 2 3 X 2 18 2 8 16 2 34 X 3 1 2 1 31 39 4 2 X 4 6 4 1 14 1 41 8 1 1 2 1 DNx 5 1 1 78 R 6 1 77 2 P 7 2 78 S

TABLE 38P Tally of 27 CDR3s of length 11 from human lambda chains Tally A C D E F G H I K L M N P Q R S T V W Y 1 4 5 6 5 4 3 X 2 3 1 2 14 5 2 Sx 3 1 7 13 6 X 4 19 2 1 1 4 DNx 5 1 4 2 2 2 1 13 2 X 6 1 3 1 21 1 S 7 1 7 12 1 4 2 X 8 2 1 10 1 6 6 1 X 9 3 1 8 10 3 1 1 X 10 1 4 1 1 1 3 1 1 6 5 3 X 11 2 25 V

TABLE 40P Synthetic Kappa light chain gene with stuffers 1 GAGGACCATt GGGCCCC                ctccgagact Scab...... EcoO109I            ApaI. 28 CTCGAG    cgca XhoI.. 38 acgcaatTAA TGTgagttag ctcactcatt aggcacccca ggcTTTACAc tttatgcttc        ..-35..         Plac                    ..-10. 98 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tc 140 acacagga aacagctatgac 160 catgatta cgCCAAGCTT TGGagccttt tttttggaga ttttcaac            PflMI.......              Hind3. M13 III signal sequence (AA seq)--------------------------->  1   2   3   4   5   6   7   8   9  10  11  12  13  14  15  M   K   K   L   L   F   A   I   P   L   V   V   P   F   Y 206 gtg aag aag ctc cta ttt gct atc ccg ctt gtc gtt ccg ttt tac --Signal-->  FR1------------------------------------------->  16  17  18  19  20  21  22  23  24  25  26  27  28  29  30   S   H   S   A   Q   S   V   L   T   Q   S   P   G   T   L 251 |agc|cat|aGT|GCA|Caa|tcc|gtc|ctt|act|caa|tct|cct|ggc|act|ctt|           ApaLI... ----- FR1 --------------------------------->|-------Stuffer->  31  32  33  34  35  36  37  38  39  40  41  42  43   S   L   S   P   G   E   R   A   T   L   S   |   | 296 |tcG|CTA|AGC|CCG|GGt|gaa|cgt|gct|acC|TTA|AGt|TAG|TAA|gct|ccc|    EspI.....                       AflII...            XmaI.... ------- Stuffer for CDR1------------------------->|- FR2 -->                                                     59  60                                                      K   P 341 |AGG|CCT|ctt|TGA|tct|                              g|aaA|CCT|  StuI...                                               SexAI... ----- FR2 ------|-----------Stuffer for CDR2---------------->  61  62  63  64  65  66   G   Q   A   P   R   |   | 363 |GGT|caG|GCG|CCg|cgt|TAA|TGA|a AGCGCT aa TGGCCA aca gtg SexAI.... KasI....             AfeI..    MscI.. Stuffer-->|--- FR3 ----------------------------------------------->  <1>  76  77  78  79  80  81  82  83  84  85  86  87  88  89  90   T   G   I   P   D   R   F   S   G   S   G   S   G   T   D 405 |act|gGG|ATC|CCG|GAC|CGt|ttc|tct|ggc|tct|ggt|tca|ggt|act|gac|       BamHI...              RsrII..... ------ FR3 ---------------------STUFFER for CDR3------------------>  91  92  93  94  95  96  97   F   T   L   T   I   S   R   |   | 450 |ttt|acc|ctt|act|att|TCT|AGA|TAA|TGA| GTTAAC TAG acc TACGTA acc tag                      XbaI...          HpaI..         SnaBI. -----------------CDR3 stuffer------------------>|-----FR4--->                                                 118 119 120                                                   F   G   Q 501                                                 |ttc|ggt|caa| -----FR4------------------->| <------- Ckappa ------------ 121 122 123 124 125 126 127   128 129 130 131 132 133 134   G   T   K   V   E   I   K     R   T   V   A   A   P   S 510 |ggt|aCC|AAG|Gtt|gaa|atc|aag| |CGT|ACG|gtt|gcc|gct|cct|agt|       StyI....                 BsiWI.. (CDR3 installed as XbaI to (StyI or BsiWI) cassette.)  135 136 137 138 139 140 141 142 143 144 145 146 147 148 149   V   F   I   F   P   P   S   D   E   Q   L   K   S   G   T 552 |gtg|ttt|atc|ttt|cct|cct|tct|gac|gaa|CAA|TTG|aag|tca|ggt|act|                                      MfeI... 866 acgcatctctaa GCGGCCGC aacaggaggag              NotI....               EagI.. A27::JH1 with all CDRs replaced by stuffers. Each stuffer contains at least one stop codon and a restriction site that will be unique within the diversity vector.

TABLE 41P Variegated DNA for kappa chains Kappa chains For CDR1: <1> ADEFGHIKLMNPQRSTVWY equimolar <2> S(0.2) ADEFGHIKLMNPQRTVWY (0.044 each) <3> Y(0.2) ADEFGHIKLMNPQRSTVW (0.044 each) <4> A(0.2) DEFGHIKLMNPQRSTVWY (0.044 each) (Ka1vg600) 5′-gct|acC|TTA|AGt|tgc|cgt|gct|tcc|cag- |<1>|gtt|<2>|<2>|  <3>|ctt|gct|tgg|tat|caa|cag|aaA|CC-3′ (Ka2vg650) 5′-caG|GCG|CCg|cgt|tta|ctt|att|tat|<1>|gct|tct|<2>|cgc|<4>|- |<1>|gGG|ATC|CCG|GAC|CGt|ttc|tct|ggt|tctcacc-3′ (Ka3vg670) 5′-gac|ttc|gct|gtt|- |tat|tat|tgC|CAa|cag|<3>|<1>|<1>|<1>|cct|<1>|act|ttc|ggt|caa|- |ggt|aCC|AAG|Gtt|g-3′

TABLE 42P Variegated DNA for lambda chains For CDR1, <1> = 0.27 T, 0.27 G, 0.027 {ADEFHIKLMNPQRSVWY} no C <2> = 0.27 D, 0.27 N, 0.027 {AEFGHIKLMPQRSTVWY} no C <3> = 0.36 Y, 0.0355{ADEFGHIKLMNPQRSTVW} no C <4> = equimolar {ADEFGHIKLMNPQRSTVWY} no C <5> = 0.36 S, 0.0355{ADEFGHIKLMNPQRTVWY} no C (Lm1vg710) 5′-gt|atc|act|att|tct|TGT|ACA|ggt|<1>|tct|tct|<2>|gtt|ggc|- |<1>|<3>|<2>|<3>|gtt|tct|tgg|tat|caa|caa|caC|CC-3′ (Lm2vg750) 5′-G|CCg|aag|ttg|atg|atc|tac|- <4>|<4>|<4>|<2>|cgt|cct|tct|ggt|gtc|agc|aat|c-3′ (Lm3vg817) 5′-gac|gag|gct|gac|tac|tat|tgt|- |<4>|<5>|<4>|<2>|<4>|tct|<4>|<4>|<4>|<4>|gtc|ttc|ggc|ggt|GGT|- |ACC|aaa|ctt|ac-3′

TABLE 43P Constant DNA for Synthetic Library CDR3 library components (Ctop25) 5′-gctctggtcaa C|TTA|AGg|gct|gag|g-3′ (CtprmA) 5′-gctctggtcaa C|TTA|AGg|gct|gag|gac- AflII... |acc|gct|gtc|tac|tac|tgc|gcc-3′ (CBprmB) [RC] 5′-|tac|ttc|gat|tac|ttg|ggc|caa|GGT|ACC|ctG|GTC|ACC|tcgctccacc-3′ BstEII... (CBot25) [RC] 5′-|GGT|ACC|ctG|GTC|ACC|tcgctccacc-3′ Kappa chains (Ka1Top610) 5′-ggtctcagtt- G|CTA|AGC|CCG|GGt|gaa|cgt|gct|acC|TTA|AGt|tgc|cgt|gct|tcc|cag-3′ (Ka1STp615) 5′-ggtctcagtt- G|CTA|AGC|CCG|GGt|g-3′ (Ka1Bot620) [RC] 5′-ctt|gct|tgg|tat|caa|cag|aaA|- CCt|GGT|caG|GCG|CC aagtcgtgtc-3′ (Ka1SB625) [RC] 5′-cct|GGT|caG|GCG|CC aagtcgtgtc-3′ (Ka2Tshort657) 5′-cacgagtcctA|CCT|GGT|- caG|GC-3′ (Ka2Tlong655) 5′-cacgagtcctA|CCT|GGT|- caG|GCG|CCg|cgt|tta|ctt|att|tat-3′ (Ka2Bshort660) [RC] 5′-|GAC|CGt|ttc|tct|ggt|tctcacc-3′ (Ka3Tlon672) 5′-gacgagtcct TCT|AGA|ttg|gaa|cct|gaa|gac|ttc|gct|gtt|- |tat|tat|tgC|CAa|c-3′ (Ka3BotL682) [RC] 5′-act|ttc|ggt|caa|- |ggt|aCC|AAG|Gtt|gaa|atc|aag| |CGT|ACG| tcacaggtgag-3′ (Ka3Bsho694) [RC] 5′-gaa|atc|aag| |CGT|ACG| tcacaggtgag-3′ (Lm1TPri75) 5′-gacgagtcct GG|TcA|CCt|GGT|-3′ (Lm1TLo715) 5′-gacgagtcct GG|TcA|CCt|GGT|- caa|agt|atc|act|att|tct|TGT|ACA|ggt-3′ (Lm1BLo724) [RC] 5′-gtt|tct|tgg|tat|caa|caa|caC|CCG|GGc|aaG|GCG|- AGA TCT  tcacaggtgag-3′ (Lm1BSh737) [RC] 5′-Gc|aaG|GCG|- AGA TCT  tcacaggtgag-3′ (Lm2TSh757) 5′-gagcagagga C|CCG|GGc|aaG|GC-3′ (Lm2TLo753) 5′-gagcagagga C|CCG|GGc|aaG|GCG|CCg|aag|ttg|atg|atc|tac|-3′ (Lm2BLo762) [RC] 5′-cgt|cct|tct|ggt|gtc|agc|aat|cgt|ttc|TCC|GGA|tcacaggtgag-3′ (Lm2BSh765) [RC] 5′-cgt|ttc|TCC|GGA|tcacaggtgag-3′ (Lm3TSh822) 5′-CTG|CAG|gct|gaa|gac|gag|gct|gac-3′ (Lm3TLo819) 5′-CTG|CAG|gct|gaa|gac|gag|gct|gac|tac|tat|tgt|-3′ (Lm3BLo825) [RC] 5′-gtc|ttc|ggc|ggt|GGT|- |ACC|aaa|ctt|act|gtc|ctc|gGT|CAA|CCT|aAG|G acacaggtgag-3′ (Lm3BSh832) [RC] 5′-c|gGT|CAA|CCT|aAG|G acacaggtgag-3′

TABLE 48P Synthtic human lambda-chain gene with stuffers in place of CDRs 1 GAGGACCATt GGGCCCC   ttactccgtgac Scab...... EcoO109I            ApaI..         -----------FR1-------------------------------------------->          1   2   3   4   5   6   7   8   9  10  11  12  13  14  15  S   A   Q   S   A   L   T   Q   P   A   S   V   S   G   S   P   G 30 aGT|GCA|Caa|tcc|gct|ctc|act|cag|cct|GCT|AGC|gtt|tcc|gGG|TcA|CCt|GGT|  ApaLI...                           NheI...          BstEII...                                                           SexAI.... ------FR1------------------> |-----stuffer for CDR1---------  16  17  18  19  20  21  22  23   Q   S   I   T   I   S   C   T 81 |caa|agt|atc|act|att|tct|TGT|ACA|tct TAG TGA ctc                          BsrGI.. -----Stuffer--------------------------->-------------------- 31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  R   S   |   |   P   |                   H   P   G   K   A 117 AGA TCT TAA TGA ccg tag                 caC|CCG|GGc|aaG|GCG| BglII                                     XmaI....    KasI.....                                           AvaI.... --|-------------Stuffer ------------------------------------->  P 150 |CCg|TAA|TGA|atc tCG TAC G                        ct|ggt|gtt| KasI....          BsiWI... -------FR3----------------------------------------------------  61  62  63  64  65  66  67  68  69  70  71  72  73  74  75   S   N   R   F   S   G   S   K   S   G   N   T   A   S   L 177 |agc|aat|cgt|ttc|TCC|GGA|tct|aaa|tcc|ggt|aat|acc|gcA|AGC|TTa|                  BspEI..          |                HindIII.                       BsaBI........(blunt) -------FR3------------->|--Stuffer-------------------------->|  76  77  78  79  80  81  82  83  84  85  86  87  88  89  90   T   I   S   G   L   Q 222 |act|atc|tct|ggt|CTG|CAG|gtt ctg tag ttc CAATTG ctt tag tga ccc                  PstI...                 MfeI.. -----Stuffer------------------------------->|---FR4---------                                                   103 104 105                                                   G   G   G 270                                                   |ggc|ggt|GGT|                                                            KpnI... -------FR4-------------->  106 107 108 109 110 111 112 113 114 115 116 117 118 119 120   T   K   L   T   V   L   G   Q   P   K   A   A   P   S   V 279 |ACC|aaa|ctt|act|gtc|ctc|gGT|CAA|CCT|aAG|Gct|gct|cct|tcc|gtt| KpnI...                      HincII..                                  Bsu36I...  121 122 123 124 125 126 127 128 129 130 131 132 133 134 135   T   L   F   P   P   S   S   E   E   L   Q   A   N   K   A 324 |act|ctc|ttc|cct|cct|agt|tct|GAA|GAG|Ctt|caa|gct|aac|aag|gct|                              SapI.....  136 137 138 139 140 141 142 143 144 145 146 147 148 149 150   T   L   V   C   L   I   S   D   F   Y   P   G   A   V   T 369 |act|ctt|gtt|tgc|tTG|ATC|Agt|gac|ttt|tat|cct|ggt|gct|gtt|act|                   BclI.... Lambda 14-7(A) 2a2 ::JH2::Clambda AA sequence tested

TABLE 50P 3-23::CDR3::JH4 Stuffers in place of CDRs                        FR1(DP47/V3-23)---------------            20  21  22  23  24  25  26  27  28  29  30             A   M   A   E   V   Q   L   L   E   S   G ctgtctgaac  CC atg gcc gaa|gtt|CAA|TTG|tta|gag|tct|ggt| Scab......  NcoI....          | MfeI  |       --------------FR1--------------------------------------------        31  32  33  34  35  36  37  38  39  40  41  42  43  44  45         G   G   L   V   Q   P   G   G   S   L   R   L   S   C   A       |ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gct|       ----FR1-------------------->|...CDR1 stuffer....|---FR2------        46  47  48  49  50  51  52  53  54  55  56  57  58  59  60         A   S   G   F   T   F   S   S   Y   A   |   |   W   V   R       |gct|TCC|GGA|ttc|act|ttc|tct|tCG|TAC|Gct|TAG|TAA|tgg|gtt|cgC|           | BspEI |                 | BsiWI|                     |BstXI.        -------FR2-------------------------------->|...CDR2 stuffer.        61  62  63  64  65  66  67  68  69  70  71  72  73  74  75         Q   A   P   G   K   G   L   E   W   V   S   |   p   r   |       |CAa|gct|ccT|GGt|aaa|ggt|ttg|gag|tgg|gtt|tct|TAA|CCT|AGG|TAG|    ...BstXI        |                                  AvrII..       .....CDR2 stuffer....................................|---FR3---       --------FR3-------------------------------------------------         91  92  93  94  95  96  97  98  99 100 101 102 103 104 105         T   I   S   R   D   N   S   K   N   T   L   Y   L   Q   M       |act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|               | XbaI  |       ---FR3-----------..> Stuffer------------->|        106 107 108 109 110         N   S   L   R   A       |aac|agC|TTA|AGg|gct|TAG TAA AGG cct TAA              |AflII |              StuI...       |----- FR4 ---(JH4)-----------------------------------------        Y   F   D   Y   W   G   Q   G   T   L   V   T   V   S   S      |tat|ttc|gat|tat|tgg|ggt|caa|GGT|ACC|ctG|GTC|ACC|gtc|tct|agt|...                                   | KpnI  |  | BstEII | 

1. A focused library of vectors or genetic packages, comprising a first set of variegated DNA molecules, each of which comprises a DNA sequence that encodes an antibody heavy chain (HC) variable region comprising a CDR1 region, a CDR2 region, and a CDR3 region, wherein the first set of variegated DNA molecules collectively encode a plurality of: (a) HC CDR1 regions selected from the group consisting of: (1) <1>₁Y₂<1>₃M₄<1>₅, wherein <1> is an equimolar mixture of each of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; (2) (S/T)₁(S/G/X)₂(S/G/X)₃Y₄Y₅W₆(S/G/X)₇, wherein (S/T) is a 1:1 mixture of S and T residues, (S/G/X) is a mixture of 0.2025 S, 0.2025, G and 0.035 of each of amino acid residues A, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, and Y; (3) V₁S₂G₃G₄S₅I₆S₇<<1>₈<1>₉<1>₁₀Y₁₁Y₁₂W₁₃<1>₁₄, wherein <1> is an equimolar mixture of each of amino acid residues A, D, E, F, G, H,. I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and (4) a mixture of any of (1) to (3) set forth above; (b) HC CDR2 regions selected from the group consisting of: (1)<2>I<2><3>SGG<1>T<1>YADSVKG, wherein <1> is an equimolar mixture of each of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; <2> is an equimolar mixture of each of amino acid residues X, R, W, V, G, and S; and <3> is an equimolar mixture of each of amino acid residues P, S, and G or an equimolar mixture of P and S; (2)<1>1<4><1><1><G><5><1><1>-<1>YADSVKG, wherein <1> is an equimolar mixture of each of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; <4> is an equimolar mixture of residues D, I, N, S, W, Y; and <5> is an equimolar mixture of residues S, G, D and N; (3)<1>I<4><1><1>G<5><1><1>YNPSLKG, wherein <1> is an equimolar mixture of each of amino acid residues A, D, E, F, G, II, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and <4> and <5> are as defined above; (4)<1>I<8>S<1><1><1>GGYY<1>YAASVKG, wherein <1> is an equimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, O, R, S, T, V, W and Y; <8> is 0.27 R and 0.027 of each of ADEFGHIKLMNPQSTVWY; and (5) a mixture of any of (1) to (4) set forth above; or (c) HC, CDR3 regions selected from the group consisting of: (1) YYCA21111YFDYWG, wherein 1 is an equimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar mixture of K and R; (2) YYCA2111111YFDYWG, wherein 1 is an equimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y: and 2 is an equimolar mixture of K and R; (3) YYCA211111111YFDAYTG, wherein 1 is an equimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar mixture of K and R; (4) YYCAR111S2S3111YFDYWG, wherein 1 is an equimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar mixture of S and G; and 3 is an equimolar mixture of Y and W; (5) YYCA2111CSG11CY1YFDYWG, wherein 1 is an equimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar mixture of K and R; (6) YYCA211S1T1FG11111YFDYWG, wherein 1 is an equimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar mixture of K and R, (7) YYCAR111YY2S3344111YFDYWG, wherein 1 is an equimolar mixture of each amino acid residues A, D, F, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; 2 is an equimolar mixture of D and S; and 3 is an equimolar mixture of S and G; (8) YYCAR1111YC2231CY111YFDYWG, wherein 1 is an equimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; 2 is an equimolar mixture of S and G; and 3 is an equimolar mixture of T, D and G; and (9) a mixture of any of (1) to (8) set forth above.
 2. The focused library according to claim 1, wherein HC CDR1s (1), (2) and (3) are present in the library in the ratio 0.80:0.17:0.02.
 3. (canceled)
 4. The focused library according to claim 1, wherein a mixture of HC CDR2s (1)/(2) (equimolar), (3) and (4) are present in the library in a ratio of 0.54:0.43:0.03.
 5. (canceled)
 6. The focused library according to claim 1, wherein 1 in one or all of HC CDR3s (1) through (8) is 0.095 of each of G and Y and 0.048 of each of A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, and W.
 7. The focused library according to claim 1, wherein HC CDR3s (1) through (8) are present in the library in the following proportions: (1) 0.10 (2) 0.14 (3) 0.25 (4) 0.13 (5) 0.13 (6) 0.11 (7) 0.04 and (8) 0.10.
 8. The focused library according to claim 1, wherein the HC CDR3s (1) through (8) are present in the library in the following proportions: (1) 0.02 (2) 0.14 (3) 0.25 (4) 0.14 (5) 0.14 (6) 0.12 (7) 0.08 and (8) 0.11.
 9. The focused library of vectors or genetic packages according to claim 1, further comprising a second set of variegated DNA molecules each of which comprises a DNA sequence that encodes an antibody kappa light chain (LC_(κ)) comprising a LC_(κ) CDR1 region, a LC_(κ) CDR2 region, and a LC_(κ) CDR3 region, wherein the second set of variegated DNA molecules collectively encode a plurality of: (a) LC_(κ) CDR1 regions selected from the group consisting of: (1) RASQ<1>V<2><2><3>LA, (2) RASQ<1>V<2><2><2><3>LA; wherein <1> is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY; <2> is 0.2 S and 0.044 of each of ADEFGHIKLMNPQRTVWY; and <3> is 0.2Y and 0.044 each of ADEFGHIKLMNPQRTVW and Y; and (3) a mixture of (1) and (2) set forth above; (b) LC_(κ) CDR2 regions <1>AS<2>R<4><1>, wherein <1> is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY; <2> is 0.2 S and 0.044 of each of ADEFGHIKLMNPQRTVWY; and <4> is 0.2 A and) 0.044 each of DEFGHIKLMNPQRSTVWY; or (c) LC_(κ) CDR3 regions selected from the group consisting of: (1) QQ<3><1><1><1>P<1>T, wherein <1> is an equimolar mixture of amino acid residues ADEFGIIIKLMNPQRSTVWY; <3> is 0.2 Y and 0.044 each of ADEFGHIKLMNPQRTVW; (2) QQ33111P, wherein 1 and 3 are as defined in (1) above; (3) QQ3211PP1T, wherein 1 and 3 are as defined in (1) above and 2 is 0.2 S and 0.044 each of ADEFGHIKLMNPQRTVWY; and (4) a mixture of any of (1) to (3) set forth above.
 10. The focused library of claim 9, wherein LC_(κ) CDR1s (1) and (2) are present in the library in a ratio of 0.68:0.32. 11-12. (canceled)
 13. The focused library according to claim 9, wherein the LC_(κ) CDR3s (1), (2) and (3) are present in the library in a ratio of 0.65:0.1:0.25.
 14. The focused library of vectors or genetic packages according to claim 1, further comprising a third set of variegated DNA molecules each of which comprises a DNA sequence that encodes an antibody lambda light chain (LC_(λ)) chain comprising a LC_(λ) CDR1 region, a LC_(λ) CDR2 region, and a LC_(λ) CDR3 region, wherein the third set of variegated DNA molecules collectively encodes a plurality of: (a) LC_(λ) CDR1 regions selected from the group consisting of: (1) TG<1>SS<2>VG<1><3><2><3>VS, wherein <1> is 0.27 T, 0.27 G and 0.027 each of ADEFHIKLMNPQRSVWY, <2> is 0.27 D, 0.27 N and 0.027 each of AEFGHIKLMPQRSTVWY, and <3> is 0.36 Y and 0.036 each of ADEFGHIKLMNPQRSTVW; (2) G<2><4>L<4><4><4><3><4><4>-; wherein <2> is as defined in (1) above and <4> is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY; and (3) a mixture of (1) and (2) set forth above, or (b) LC_(λ) CDR2 regions <4><4><4><2>RPS, wherein <2> is 0.27 D, 0.27 N, and 0.027 each of AEFGHIKLMPQRSTVWY and <4> is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVW, or (c) LC_(λ) CDR3 regions selected from the group consisting of: (1)<4><5><4><2><4>S<4><4><4>-<4>V, wherein <2> is 0.27 D, 0.27 N, and 0.027 each of AEFGHIKLMPQRSTVWY; <4> is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVW; and <5> is 0.36 S and 0.0355 each of ADEFGHIKLMNPQRTVWY; (2)<5>SY<1><5>S<5><1><4>V, wherein <1> is an equimolar mixture of ADEFGHIKLMNPQRSTVWY; and <4> and <5> are as defined in (1) above; and (3) a mixture of (1) and (2) set forth above.
 15. The focused library according to claim 14, where LC_(λ) CDR1s (1) and (2) are present in the library in a ratio of 0.67:0.33. 16-17. (canceled)
 18. The focused library according to claim 14, wherein LC_(λ) CDR3s (1) and (2) are present in the library in an equimolar mixture.
 19. The focused library according to claim 1, wherein the first set of variegated DNA molecules collectively encode a plurality of HC CDR1s set forth in (a), HC CDR2s set forth in (b), and HC CDR3s set forth in (c).
 20. (canceled)
 21. The focused library according to claim 9, wherein the second set of variegated DNA molecules collectively encode a plurality of LC_(κ) CDR1s set forth in (a), LC_(κ) CDR2s set forth in (b), and LC_(κ) CDR3s set forth in (c). 22-43. (canceled)
 44. The focused library according to claim 14, wherein the third set of variegated DNA molecules collectively encode a plurality of LC_(λ) CDR1s set forth in (a), LC_(λ) CDR2s set forth in (b), and LC_(λ) CDR3s set forth in (c).
 45. The library of vectors or genetic packages according to claim 1, wherein the library is a library of vectors.
 46. The library of vectors or genetic packages according to claim 45, wherein the vectors are yeast vectors.
 47. The library of vectors or genetic packages according to claim 1, wherein the library is a library of genetic packages.
 48. The library of vectors or genetic packages according to claim 47, wherein the genetic packages are cells, spores, or viral particles.
 49. The library of vectors or genetic packages according to claim 48, wherein the genetic packages are phage particles.
 50. The library of vectors or genetic packages according to claim 48, wherein the genetic packages are yeast cells.
 51. The library of vectors or genetic packages according to claim 50, wherein the yeast cells display the antibody heavy chains encoded by the first set of variegated DNA molecules. 